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METABOLISM
OF
LACTIC
ACID
BACTERIA
A the sis presented in p a rtial fulfilment of the requirements
for the de gree of Doctor of Philosophy in Biochemistry at
Massey Unive r s ity, New Zealand .
Terence
David
THOMAS
1968
M
Alil ifillillIlifllTl llllliiif
1061883881
Y
METABOLISM
OF
LACTIC
ACID
BACTERIA
A the sis presented in p artial fulfilment of the requirements
for the de gree of Doctor of Philosophy in Biochemistry at
Massey Unive r s ity , New Zealand .
Terence
David
1968
THOMAS
N.. .
ABSTRACT
Streptoc occus l a ctis organisms were grown in l actose­
l imite d batch culture and the survival characteristics of
washe d organi sms were examine d at the growth temperature .
Washe d suspension s had high initial viabilitie s ()9 9 % ) which
were maintaine d for varying pe riods depending on the
presence of certain added materials in the buffer and the
2+
conditions o f incub ation .
Adde d Mg
marke dly prolonge d
surviva l , while high bacterial concentration s a l s o extende d
2+
survival time s , probably be cause Mg
was excrete d by the
bacteria .
Surviving organisms in some conditions showe d
2+
prolonged division l ags , e specially in the absence of Mg .
Addition of trace amounts of EDTA de crea se d the death rate
by removing toxic cation impurities , while the buffer salt
concentration had l ittle e ffe ct on survival within wide
l imits .
The optimum pH value for survival was ne ar 7 . 0
and s urvival time s increased considerably at l owe r
temperature s .
Agitation and aeration tende d to decrease
survival and the de ath rate was not influence d by the phase
of growth at which the organisms were harve ste d from a
lactose-limiting me dium .
Addition of c a s amino acids incre a se d survival times
. .
2+
a rglnlne was a 1mos t as
. the presence of Mg
mark e dl y ln
e ffe ctive as the c omplete mixture of amino acids while
other individual amino acids teste d gave only slight
incre a se s in survival time s .
Fermentable carbohydrates
accelerate d de ath of starve d organisms irrespe ctive of the
"
growth phase from which they were harve sted and of the
l imiting nutrient; the a ccele rate d death rate was re duce d
2+
by a dditi on of Mg .
Glucose metabolism procee de d at a
much faster rate than arginine metabo li sm , the oretically
producing about 7 . 5 time s as much ATP .
This rapid
generation of ATP may be responsible for the more rapid
iii .
death rate s with a dde d carb ohydrate s . Arginine substantially
re duce d the lethal e ffect of a dverse pH value s and suppressed
the leakage of free intracellular amin o acids into the exte rnal
me dium .
Survival studies were followe d by an inve stigation o f the
change s which took place in starve d or ganisms and their
relation to surviva l .
N o polyglucose or p oly- � -hydroxybutyrat
was dete cte d and starve d organisms had a negligible respiration
rate .
Soluble prote in was re lease d from viable organisms. into
the suspending buffer and the intracellular free amino acid
pool decline d stea dily with the components appearing in the
suspending buffer; a net increase in the total amount of free
amin o acid indicate d s ome protein hydrolysis .
Chloramphenicol
reduced the death rate s in s ome environments , possib ly by
s uppressing prote in de gradation .
RNA was hydrolyse d with the
rele ase o f u . v . -absorbing b a s e s and ribose from the organisms .
Conditions which promote d rapid RNA breakdown also produce d
rapid death rate s and l ong cell divis ion lags in surviving
The re was n o appreciable de gradation of
o rgani sms .
carbohydrate or DNA .
Afte r 2 8 hr . starvation in buffer
2+
c ontaining Mg , the b acterial dry wt . de crea se d by 2 6%; loss
o f RNA, prote in and free amino acids accounte d for 1 0 . 3 % , 7 . 3%
The products of
and 2 . 7% of the total bacterial mas s loss .
polymer hydrolysis appeare d to be released in an unde gra de d
form into the external buffer and the re was n o appreciable
f ormation o f l actate , ammonia or volatile fatty acids possibly
indicating the absence of any important endogenous energy
s ource s .
Prote in synthe sis , determine d by the incorporation of
14
valine_ C into TCA-insol uble materia l , was barely detectable
2+
when organi sms we re starve d in buffer containing Mg .
Addition o f an ene rgy s ource allowe d l imite d prote in
synthe sis while glucose produce d a much highe r rate of
14
val ine_ C uptake and incorporation than arginine .
iv .
Although arginine prolonge d survival this was not due to the
limite d prote in synthe sis which took place .
The survival
capa city of starve d organisms could be corre late d with the
ability to synthe size prote in which in turn may be corre l ate d
with RNA stability .
A new method was de ve lope d for the a ss ay of glycolytic
activity in microorganisms.
Organisms we re incub ate d with
4
1
Samples
glucose -U- C and s amples remove d at inte rvals .
were chromatographe d on DBAE-cellulose paper strips in
de ionize d wate r which separate d the r adioactive anionic
p roducts of glycolysis ( l a ctate , acetate and formate) from
the unfermente d gluco se .
The activity o f the two fra ctions
was then determine d by li qui d scintillation counting .
T he
glycolytic activity of starve d organisms de cline d steadily
and was not corre late d with survival .
Phospholipid was broken down on prolonge d starvation and
the perme ability prope rties of the organism we re gra dually
lost .
Addition of spermine gave enhance d survival and
suppre ssed the re lease of u . v . -absorbing mate rial .
Lactic
dehydrogenase and DNA were re lease d as the death r ate
+
incre ased in buffer conta ining Mg2 and e ventually, well
afte r de ath, cell lysis occurre d .
E l e ctron micrographs
indicated that addition of amino acids ma intaine d cell
structure s for a much longe r pe riod and in this system cell
lysis occurre d a s the death rate incre a se d .
It was con-
clude d that the de ath rate of starve d S . lactis organisms
in phosphate buffer was partly dependent on the pre sence
+
of Mg2 , which probably acted by promoting polymer stability,
particularly that of RNA .
In this envi ronment , a suitable
exogenous ene rgy source furthe r enhance d survival which may
ultimate ly be a function of ce ll wall and membrane stability .
v.
ACKNOWLEDGEMENT S
The author is ind�bte d to the New Ze aland Dairy
Re search Institute for providing the opportunity to
undertake this inve stigation .
P a rticul ar thanks a re extende d to:
P rofe ssor R. D . Batt an d Dr. W . A . McGillivray for the ir
a dvice and encouragement throughout the course
of this work ;
Dn R. C . Lawrence and Mr. J . G . Robe rtson for he lpful
comments on the manus cript ;
Mrs. P . Lyttleton and Mr. K . I . Williamson for preparing
the ele ctron micrographs ;
Dr. C . R . Boswell for assisting with atomic absorption
spe ctrometry me asurements ;
Mr. J . G . Dingle for assisting with amino acid analyse s ;
Mr . R . V . Toms for the reproduction of figure s and plate s ;
Mrs . J . Crompton and Mrs. R. V . Haggett fo r typing the
manuscript .
vi .
PREFACE
A maj or function of the Mi crobiology De partment o f
the New Zealand Dairy Re search Institute is the mainten­
ance of active culture s of various lactic acid b acte ria
for use in the che e se industry .
This work is vital
for the e fficient functioning o f one of the country' s
maj or industrie s .
The most important che e se ' starter'
organisms are the lactic streptococci .
This group
consists of Streptococcus lactis , Streptococcus cremoris
and variants .
No detaile d me asurements have been
reported on factors affe cting the survival and a ctivity
of lactic streptococc i , or closely relate d b acte r i a , at
growth tempe rature s .
Accordingly, it was de cide d t o
unde rtake such a n inve stigation which c ould possibly
produce re sults of cons iderable practical significance .
The Introduction i s d�vide d into two parts .
P art I contains a lite rature review on the metabolism
of lactic acid b acte ria which provide s a b a ckground
for subse quent discussions .
P art I I contains a summary
of the reports most rele vant to studie s on the survival
of S . lactis .
Publ ications t o date from re sults presente d in
this the sis are entitle d:
' Survival of Streptococcus lactis in starvati on
conditionS , I. gen . Microbiol . , iQ , 3 6 7, ( 1 9 6 8 ) ,
' A new method for the assay o f glycolytic a ctivity with
spe cial refe rence to microorganisms' , Anal . Biochem . ,
in Press .
vii .
CONTENTS
INT RODUCTION
PART I .
Page
THE METABOLISM OF LACTIC ACID BACTERIA
WITH SPECIAL REFERENCE TO STREP TOCOCCUS
Gene ral characte ristics
1
1
Carbohydr ate fe rmentation
3
LACTIS
(i)
( ii )
( iii )
( iv )
(v)
PART I I .
Metabolism of non-carbohydrate
compounds
Growth re quirements
Summary
SURVIVAL OF VEGETATIVE MICROORGANISMS
(i)
( ii )
( iii )
9
11
12
13
Definition of terms
13
Methodology
13
Re sults o f viability me a surements on
microorganisms starve d at the
growth temperature unde r ' minimum­
( iv )
(v)
(a)
(b )
(c)
stre s s ' conditions
Effect of ionic environment
Effe ct o f e xogenous substrate s
Effe ct of b a cterial density
17
17
19
21
Catabolism and turnove r o f cell
constituents in viable , starve d
bacte ria
23
The role of endogenous met abolism in
the survival o f starve d b a cteria
28
AIMS OF THE PRESENT INVESTIGAT ION
35
METHODS
37
Glycolyti c a ctivity a ssay
37
45
54
Ele ctron micro s copy
62
Microbiological p roce dure s
Analytical metho ds
viii .
P age
64
EXPE RIMEN TAL
PART
1.
SURVIVAL O F STREPTOCOCCUS LACTIS
Growth o f the organism
Bacte rial numbers in re suspende d systems
Survival in re suspende d systems
Effe ct of:
EDTA
divalent metal ion s
bacte rial concentration
growth phase and me dia compo sition
salt concentration
pH value
tempe rature
atmosphere and agitation
metabolic inhibitors
adde d carbohydrate s
adde d amino acids and other growth
64
64
66
70
70
70
71
72
72
73
74
75
75
77
77
me dium components
PART II .
Growth characte ristics of survivors
79
CHANGES I N VIABLE ORGANISMS I N STARVATION
79
CONDITIONS
(i)
Chemical studie s
Oxygen uptake studies
79
79
80
Change s in b acte rial protein and
80
' Re se rve' polyme rs
t otal nitrogen
Re lease of amino a cids and ammonia
Change s in nucle ic acids
Change s in carb ohydrate
( ii )
( iii )
( iv )
Change s in l ipids
83
87
90
90
P rote in synthe sis in starve d S . lactis 9 5
Metabolism of arginine and glucose
100
Change s in pe rme abil ity and ultra-
102
structure
Me asurement of b acte rial lysis
102
ix .
Page
Effect of spermine
General ultra structural f eatur es of
1 05
1 05
the c ell
Ultra st ructural changes in sta rved
1 06
cells
DI SCUSSION
Survival o f starved bacteria
Survi val measur ement
Eff ect o f :
Mg
2+
108
108
108
109
bacterial density
109
added substrates
III
temperature and pH
Changes in starved organi sms
Po lymer d egra dation
Metabolism of arginine and gluco s e
Changes i n p ermeability and
III
112
116
117
1 21
ultrastructur e
Conclusions
REFERENCES
122
124
INTRO DUCTION
P ART I .
THE METABO LISM OF LACTIC ACI D BACTERIA WITH SPECIAL
REFERENCE TO STREP TOCOCCUS LACTIS
i)
GENERAL CHARACTERISTICS
The lactic acid b a cteria are members of a s ingle family,
the Lactob a cillace ae , the spe cie s of which ferment glucose
with the pre dominant formation of lactic acid .
The genus
Streptococcus ( Table 1 ) contains the large st and most varie d
group of lactic acid bacteri a .
The best known spe cie s in this
genus are r athe r spe cialize d ecologically as a re sult of the ir
e xacting nutritional requirements and strong fermentative
metabolism
.
I
The natural habitat of Streptococcus lactis
was thought to be on the surface of plants ( Stark & She rman ,
1 9 3 5 ) , whe re the organism grows on se crete d plant materials .
Following the dome stication of lactating animal s , lactic
streptococci have a dapted to a milk environment and the se
organisms gene rally pre dominate in raw milk which has turne d
sour ( Orl a-Jensen , 1 9 4 2 ) .
Members of the family Lactobacillaceae are non-spore
forming and with a few exception s , they a re non-motile .
They a re strongly Gram-po sitive in young culture s but often
appe ar Gram-ne gative in old culture s .
The organisms are
generally catalase -ne gati ve ( see Whittenbury , 1 9 6 4 ) and
microae rophilic or facultative ae robe s .
The lactic group
of streptococci ( T able 1 ) consist of closely relate d
organisms .
Stra ins of S . lactis grow mainly as dipl ococci
or in short chains while those of Streptococcus cremoris
ten d t o form l ong chain s , the morphology being dete rmine d
l argely by growth conditions .
Both organisms occur a s
ovoid c e l l s e l ongate d i n the dire ction o f the chain with
individual cells 0 . 5 - 1 . 0� in diamete r .
Most stra ins of
S . lactis produce the antibiotic nisin which i s cons idered
2.
T able 1.
Classification of lactic aci d b acteria ( Be rgey , 1 9 5 7).
O rde r
Eubacteriales
F amily
Lactobaci llace ae
Tribe
I Streptoc occeae
I I Lactobacilleae
Peptostreptococcus
Leuconostoc
Lactobacillus etc
Pediococcus
Diploc occus
Streptoc occus
pyogenic gp
viridans gp
ente r oc occus gp
l actic gp
l actis, cremoris
( di acetilactis , the rmoph ilus)
3.
by Hurst ( 1 966 , 1 96 7) t o be involve d in cell regulation ,
particularly in the proce sses connected with the initiation
and cessation of growth.
Many strains of lactic acid b acteria are of c onside rable
industrial and a gricultural importance , primarily because of
the ir fermentative activit ie s .
Applications in which they a re
involve d include ( a ) the preparation of a wide range of
fermented milk products , ( b ) curing of me ats , ( c ) manufacture
of l actic aci d and ( d ) the fermentation of vegetable and othe r
plant products.
The l actic streptococci are of particular
importance in the manufacture of cheese because of their
cap ac ity f o r c onsistent , rapid, l actic acid production and
the ir ability to impart de s irable flavour and texture to the
final product.
Lactic acid bacteria a re also involve d in
the spoilage of food products.
Few biochemical inve stigations have been undertaken
with S . lactis ( see re views by Marth , 1 96 2 ;
Moller-Madsen , 1 96 3) .
Reiter &
This may be a re sult of the restricte d
·nature o f the ir metab ol ism and the diff iculty in maintaining
experimental cultures with reproducible activitie s .
The bulk
of the published work on lactic streptococci conce rns microbiol­
ogical a spects of the i r use a s chee s e ' starte r ' organisms.
As
a c onse quence of this emphasis , the re are c onside rable gaps in
our basic knowle dge of the ir metabolism .
The following
discussion de als with a spects of the metabolism of s ome closely
relate d species of microorganisms .
ii )
CARBOHYDRATE FERMENTATION
The ability of l actic acid bacte ria to ferment carbo­
hydrate s other than glucose varies wide ly and the se differences
a re often use d in microbial classification .
Lactic streptoc occi
c an utilize glucose , galactose , lactose , mannose and fructose
4.
a s ene rgy source s for growth (Sandine , Ellike r & Hays , 1 9 6 2) .
Many strains , especially o f S . cremoris , are unable to fe rment
maltose , p re sumably be cause of their inability to induce the
appropriate spe cific transport or de gra dative en zyme s ( Citt i ,
Sandine & Ellike r , 1 9 6 7) .
The � -gala ctosidase of S . lactis
has been shown to have similar propertie s to that of
Es cherichia coli ( Citt i , Sandine & Ellike r , 1 9 6 5) .
Fe rment­
ation of pentose s and othe r carbohydrate s is restricted to
only a few strains of lactic streptococci (Sandine et al . ,
1 9 6 2) .
T he re are no reports of ribose f e rmentation by
S . lactis .
Two metabolic groups were recognized among lactic acid
b a cteria by O rla-Jensen ( 1 9 1 9) .
The homofermentative or
homolactic group was characterized by the formation of mainly
lactic acid from glucose fermentation .
This group include d
all members of the gene ra Streptococcus , Diplococcus , P e diococcus
and most members of the genus La ctobacillus .
The second group ,
the hete rofermentative lactic acid b a cte ria , produced consider­
able amounts of CO , ethanol or a cetic acid, in a ddition to
2
lactic acid ( for re views see Wiken , 1 9 5 9 ; Wood, 1 9 6 1 ;
Re iter & Molle r-Ma dsen, 1 9 6 3) .
This group include d all
members of the gene r a Leuconostoc and P eptostreptococcus
( Be rgey , 1 9 5 7) .
Early inve stigations involving kineti c , inhibitor and
enzyme studie s , pro vi de d e vidence that the pathway for homo­
lactic glucose fe rmentation was essentially the same as that
found in muscle glycolysis or in ye ast f e rmentation .
I sotopic studies with specifically labelle d glucose_
14
C
( Gibb s , Dumro s e , Bennett & Bubeck, 1 9 5 0) confirme d the
pre sence of the Emb den-Meye rhof pathway of glycolysis in
homofermentative lactic acid b acteria .
T he se studies
demonstrate d that the glucose molecule was split symetrically
into two 3-carb on units, lea ding to a de f ined distribution
of the glucose carb on among the carbon atoms of the ferment­
ation products ( Fig . 1) .
Gibbs et a l. , ( 1 9 50) demonstrated
that unde r b oth anaerobic and aerobic conditions,
14
Lactobacillus casei fermented glucose_ 1_ C a lmost exclusive­
ly to methyl labelled l actate with a 5 0 % dilution of the
specific activity in the methyl group compared with that
14
of carb on atom 1 of glucose.
When glucose' _ 3, 4_ C wa s
fe rment e d, carb oxy-labe l le d l actate was forme d without
dilution of the specific activity .
No fixation of
labelled CO
occurred during fermentation.
The distribut.,..
2
ion of carb on atoms.in the f inal products is consistent
with the ope ration of the Emb den-Meyerhof glycolytic
pathway ( Fig. 1) .
Thi s pathway is assumed to operate in all h omolactic
b acteria, a lthough it has been conclusively demonstrated
in only a few species.
Direct evidence for the involve­
ment of this pathway in S . l actis is limited to brief
reports of the presence of s ome of the glycolytic en zyme s
in cell-free extracts (Buyze, Hamer & Haan, 1 9 5 7 ;
1960;
Shahani & Vakil, 1 9 62) .
Shahani,
The latter authors have
not reporte d any expe rimental details.
It is well recogni ze d that products othe r than lactic
acid may re sult from glucose fermentation by h omolactic
organisms, a lthough these products are formed in small
amounts only .
Smal l quantities of formate, acetate,
propionate, butyrate, eth anol, CO , 2, 3 -but ane diol,
2
acetoin, diacetyl and acetaldehyde have been reported as
gluc ose fermentation products of such o rganisms ( Hammer,
1 920 ;
Foster, 1 92 1 ;
1958 ;
Harvey, 1 9 6 0 ) .
Langwill, 1 924 ;
P latt & Foster,
Although none of these authors
demonstrate d that the end products actually a rose from
glucosej the oxidation-reduction indices and the carbon
1 CO
2 CH
3
I
CH
1
2
0H
4 COOH
I
I
CH
I
I
C
I
C
I
C
I
1 C
I
2 C
3
2
CHO
<
3
4
)
2 C
!
3 OOH
5
6
6 C
6 C
gluco s e
pyruvat e
<
H et erola cti c
f ermentation ( HMP)
Fig . 1 .
I
l � 1 , 6 CHI
2 , 5 CHOH
4 COOH ) "
...
mlnor
I
5 C
'- ,
5 CHOH
3
3 , 4 COOH
I
)
�
3 , 4 CO
3
2
or H COOH
2 , 5 CH 0H
2
COOH
1 , 6 CH
CH
I
3
I
3
Homo 1 a ctic
f ermentation ( EMP )
Di stribution o f gluco s e ca rbon in th e maj or
f ermentation products o f la cti c acid bacteria ( Bus s e , 1 9 6 6 ) .
6.
recoveries reported by P latt & Foster ( 1 9 5 8) were s o close to
those expecte d the oretical ly, that it seems like ly that the
wide variety of end product s were forme d from glucose rathe r
than from other carb on s ources i n the me dium .
P l att &
Foster ( 1 9 5 8) mea sure d anaerobic glucose fermentation
balance s for seven typical homofermentative streptococci.
The c omb ine d fe rmentation products othe r than l actate
( acetate, formate, CO , eth anol, glycerol, diacetyl, acetoin
2
and 2, 3 -butanediol) accounte d f or less than 1 8% of the
recove re d carbon in a ll culture s.
For S . l actis and
S . cremoris these product s amounte d t o about 5% of the
recovered carb on at pH 7 . 0 .
P l att & Foster ( 1 9 5 8)
suggeste d that the s e c ompounds were produce d by glycolytic
reactions but the e quimolar relationship of one- and two­
carb on products t o be expecte d from pyruvat e was not
obse rve d experimentally.
White & She rman ( 1 94 3) examine d the effect o f
aeration on glucose fermentation by a range of streptoc occi.
While little difference was observe d between anaerobic and
1
normal 1 aerobic c onditions, vigorous aeration marke dly
inhibite d growth and in all cases lactic acid was produce d
in re duced amounts .
Gunsul a s & Niven ( 1 94 2) found that
the production of formate, acetate and ethanol by
Streptoc occus ligue f aciens was marke dly increased at
a lkaline pH at the expense of lactate .
By contrast,
White, Steele & P ie rce ( 1 9 5 5) reported no ma rke d change
in the relative amounts of glucose fermentation products
of Streptococcus py ogenes o ve r the pH range 6 . 0 -7. 8.
With
this organism, galactose fermentation resulte d in only
2 5- 5 7% conve rsion to l actat e, while glucose fermentation
re sulted in 9 0 -9 5% c onve rsion t o lactate .
According t o
Shahani & Vakil ( 1 9 6 2), a transition of S. l actis from a
h omofe rmentative t o a hete rofermentative type as a result
of changing glucose for another carbohydrate is a
7.
cha racteristic finding .
Howe ve r, S . l actis is known to
ferment ga lactose via the Embden-Meye rh of pathway ( Kandle r,
1 9 6 1) .
Subs equently, Fukuyama & OIKane ( 1 9 62) demonstrated
that Streptococcus f aecalis also fermente d ga lactose via
the Embden-Meyerhof pathway, so presumably the a lteration
of end products occurs at the pyruvate level .
Various en zymes of the hexose monophosphate (HMP)
pathway have been demonstrate d in cell-free extracts of
S . l actis by Buyze et ale ( 1 9 5 7) .
Busse ( 1 9 6 3) f ound
14
C02
that f our strains of S. lactis produced much more
14
14
from glucose- 1_ C than from glucose_ 6_ C, sugge sting
that as we ll as h omolactic fermentation, s ome of the
glucose was oxidi ze d to gluconate and then decarboxylated
to pentose ( se e re view by Bus se, 1 9 6 6).
It has also been
sugge ste d that S . l actis may be capable of fermenting
glucose via the·Entne r-Doudoroff pathway (Re ite r & Moller­
Madsen, 1 9 6 3) .
There is h owe ve r, no expe rimental
e vidence for this type of metabolism and the Embden-Meyerhof
glycolytic pathway is undoubte dly the quantitatively
significant pathway in lact ic streptococci .
Until 1 9 5 0, the Emb den-Meyerhof pathway was be lieve d
to b e the only r oute for the dissimilation of carbohydrates .
Howe ve r DeMoss, Bard & Gunsalus ( 1 9 5 1) f ound that
Leuc onostoc mesente roide s produce d CO , ethanol and lactic
2
acid in a c onstant ratio of 1: 1: 1 which is not consistent
with the glyc olytic pathway .
Furthermore, it was found
that the en zyme s aldolase and triose pho sphate isome rase
we re absent in L . mesenteroides and the ratio of products
could not be var ie d significantly by changing the pH of
the culture .
I s otopic studies by Gunsalus & Gibbs ( 1 9 52)
c onfirme d the existence of an alternative pathway.
14
Fe rmentation of gluc ose- 1_ C by L. me senteroide s re sulte d
in all the isotope be ing f ound in the CO , whereas with
2
8.
glucose- 3, 4-
14
C, the isotope wa s containe d in carb on 2
of the ethanol and the carboxyl carb on of the l actate
( Fig. 1 ) .
The se findings we re later c onfirme d and
extende d to elucidate the c omplete HMP pathway for this
organism ( se e re view by Woo d, 1 9 6 1) .
The re lease of glucose carb on 1 as CO
is character­
2
istic of glucose fermentation by the HMP p athway (Gibbs,
Sokatch & Gunsalus, 1 9 5 5).
By the a ssymetric bre akdown
of the glucose molecule, the various products of hetero­
lactic fermentation a re not formed by side reactions as
was the case in the glyc olytic pathway .
Homolactic
fermentation results in isotope dilut ion, while in hete ro­
lactic fermentation no dilution occurs ( Fig. 1) .
Pre sence of the Entner-Doudoroff pathway (Entner &
Doudoroff, 1 9 52), which include s the formation of
2-ket o- 3-de oxy- 6-phosphogluconate from gluco se, has
been demonstrate d in So faeca lis ( Sokatch & Gunsalus,
1 9 5 7) but this r oute appe ars to be of limite d significance
in lactic acid b acteria.
Similarly, the fructose-6-
phosphate pathway is not common in the se organisms,
although Lactobacillus bifidus ferments glucose to l actate
and acetate in a ratio of about 1:2 ( Scardovi, 1 9 64 ;
Scardovi & Trovatelli, 1 9 6 5), a s the oretically require d
by this route.
The ability of an organism to produce enzymes of
the Embden-Meyerhof and HMP pathways has been sugge ste d as
a ba sis for the diffe rentiation of the genus Streptococcus
from Leuconostoc and for the separation of lactic acid
b acteria into homo- and heterofermentative groups ( Buyze
et a l., 1 9 5 7).
As a re sult of the en zymic a ssays of a
large number of lactic acid b acteria, Buyze et a lo (1 9 5 7)
c onclude d that three fermentative types of lactic acid
9.
bacteria exist:
( a) obligate h omofermenters possessing
a ldolase but l acking dehydrogenases for glucose- 6-ph osphate
and 6-ph osphogluconate ;
( b) obli gate heterofermenters
possessing b oth the se dehydrogenases but l acking aldolase ;
(c) facultative h omofermenters possessing a ldolase, as
we ll as the C -dehydrogena ses but gene rally metab olizing
6
ca rbohydrate by means of the Emb den-Meye rhof glycolytic
pathway .
S . lactis appe ars to be long to the last group,
the total and relative amounts of by-products be ing
dete rmined by the structure of the carbohydrate, the
strain and growth c onditions of the organism, a numbe r
of environmental factors such as pH, the presence or
absence of oxygen and CO , and inhe rent factors such as
2
change s in the metab olic state of the organism during
c ontinue d cultivation .
From the info rmation now a va ilable,
it seems that the terms homo- and heterofermentative may
have outlive d their usefulne ss in de scrib ing the carbo­
hydrate metab olism of lactic acid bacteria .
A fuller
de scription of enzyme activities and fermentation products
woul d be required t o provide a satisfactbry grouping of the
main types of lactic acid b acteria .
iii)
METABO LISM OF NON-CARBOHYDRATE COMPOUNDS
Cert a in species of enteroc occi in the genus
Streptococcus, are able to met abolize pyruvate, citrate,
ma late, glycerol or gluconate alone as s ources of ene rgy
for anaerobic growth ( se e re view by De ibe l, 1 9 64) .
Fermentation of citrate by l actic acid bacte ria involve s
the splitting t o acetate a n d oxalacetate, decarboxylation
of oxalacetate to pyruvate and conve rsion of pyruvate to
lactate or to acetoin and CO 2 (Kandle r, 1 9 6 1) . Streptococcus
diacetilactis was unable to use citrate as a s ource of
energy for growth but the addition of citrate to a lactose
c onta ining medium increased the specific growth rate by 3 5%
(Harvey & Collins, 1 9 6 3) .
These authors suggested that
10 .
citrate fermentation provide d a carbon s ource for e ssential
cell c onstituents which we re synthe sized at a aowe r rate in
the ab sence of citrate , all excess pyruvate being c onverte d
to acetoin .
Citrate uptake by S . diacetilactis is
en zymically me diated by an energy dependent , inducible
transport system (Harvey & Collins , 1 9 62) , which i s absent
from the other lactic streptoc occi .
Apart from carb ohydrate , arginine is the only
substrate reported from which S . lactis can obtain ene rgy
(see Barke r , 1 9 6 1) .
The catabolism of arginine by S . lactis
was first demonstrate d by Niven, Smiley & Sherman ( 1 942)
and the enzyme s catalysing the se reactions we re subsequently
characterized by Kor zenovsky & Werkman ( 1 9 5 3 , 1 9 54) .
Arginine is c onverted to ornithine , ammonia and CO2 ,
probably by the following reactions (Barke r , 1 9 6 1):
arginine
+
H 0
2
+
citrulline
P
carbamyl�P
+
�
-------->�
i
ADP
>
citrulline
+
ornithine
+
+
+
NH
3
CO
2
NH
3
carbamyl�P (NH . CO . OP 0 H�
2
3
ATP
Arginine is catabolized by the s ame pathway in S . f aecalis
but the ene rgy produce d did.not permit growth in a define d
me dium (Bauchop & El s den , 1 9 6 0) .
The p roduction of carbonyls and othe r volatile
c ompounds by l actic streptoc occi has receive d much attention
in recent ye ars b ecause of the possible role of the se
compounds in flavour enhancement or flavour de fects in
dairy product s (see re views by Mabbitt , 1 9 6 1 ;
Ve damuthu, Sandine & Ellike r , 1 9 6 6a , b) .
Marth , 1 9 6 3 ;
The formation of
some of the se products from secondary reactions of pyruvate
during ca rbohydrate or citrate fermentation has already
been discus se d .
Ce rtain strains of l actic streptoc occi
can also produce volatile carb onyl compounds and fatty
acids from s ome amino acids , a lthough normally in trace
amounts only (MacLe o d & Morgan , 1 9 5 5 , 1 9 5 6 , 1 9 58 ;
N akae
11.
& Elliott, 1 9 6 5 a , b ) .
For example, it h a s been reporte d
that Streptococcus l actis va r . maltigene s pro duces
3 -methylbutanal, 2-methylbutana l, 2-methylpropanal,
3 -methylthiopropanal and phenyl acetaldehyde by transamination
followe d by decarboxylation of the
�
-keto acids forme d from
leucine, isoleucine, val ine, methionine and phenyla lanine
re spectively ( MacLe od & Morgan, 1 9 5 8 ) .
The re is no e vidence for the ope ration of a
tricarboxylic acid cycle in l actic aci d bacte ria or for
the pre sence o f cyt ochrome s but several spec ies can actively
respire through a flavoprote in me diate d hydrogen transport
system ( see re views by Gunsalus, 1 9 5 8 ;
iv)
Dolin, 1 9 6 1 ) .
GROWTH REQUI REMENTS
Some specie s of enteroc occi can grow with ammonium
i on as the sole nitrogen s ource and a non�carbohydrate
ene rgy substrate ( Diebel, 1 9 64 ) .
Howe ve r, all strains of
l actic streptoc occ i re quire f o r growth, a carb ohydrate,
amino acids, vitamins and inorganic salts ( se e re view by
Re ite r & Moller-Ma dsen, 1 9 6 3 ) .
Re ite r & Oram ( 1 9 62 ) use d
a synthetic medium in the ir .studie s and determine d the
essential growth re quirements of S . l actis and S . cremoris
by the omission of individual sub stances from the growth
me dium .
The following amino ac ids we re f ound to be ( a )
essential:
valine, glutamic acid, methionine, leuc ine,
isoleucine, histidine and arginine ;
aspartic acid and cystine ;
some stra ins:
and tryptophan .
( b ) non-e s sential:
(c ) occ a s i onally re quire d by
alanine, phenyl alanine, glycine, thre onine
The re quirement for proline, lysine,
serine and tryosine by most strains of S . cremoris, helps
to distinguish this organism from S . l act i s .
Essential
vitamins for the growth o f l actic streptoc occ i were
nicotinic ac id, pantothenic acid and b i ot in ;
pyridoxal
stimul ate d growth while s ome stra ins re quire d riboflavin
and thiamin .
Vitamins or amino ac ids may be e ssential,
12 .
stimulatory o r not r e quired, depending on the extent of
def iciencies or imbalance s in the basal medium ( see Reiter &
Molle r-Ma dsen, 1 9 6 3) .
This nutritional data is consistent
with earlier f indings of Niven ( 1 944) and Anderson &
Elliker ( 1 9 5 3) .
Other growth f actors have been shown to be
e s sential or stimulatory for the growth of S. lactis under
certain conditions .
The se include peptides ( Ga rvie &
Mabbitt, 1 9 5 6), nucleic acid bases (Sne ll & Mitchell,
1 94 1 ;
Koburger, Speck & Aurand, 1 9 6 3), acetate and oleate
( Collins, Nelson & P a rme lee, 1 9 5 0) and r(-lipoic acid
(Re iter & Oram, 1 9 62) .
v)
SUMMAR Y
S . lactis was shown to c onta in the en zyme s aldola se,
glucose-6-ph osphate dehydrogenase and 6-phosphogluc onate
dehydrogena se ( Buyze et a le 1 9 5 7) , indicating the pre sence
of b oth the Embden-Meyerhof and the
carb ohydrate fermentation.
HMP
pathways f or
The end products of glucose
fermentation by S. lacti s were consistent with fermentation
by the Embden-Meyerhof glyc olytic pathway ( P latt & Foster,
1 9 5 8) and this is normal ly the main fermentation r oute.
Howe ve r, variation in carb ohydrate structure and environmental conditions may vary fermentation patterns .
All stra ins
of S . lactis are capable of fermenting glucose, galact ose,
lactose, mannose, fructose and maltose ( Sandine et a l. ,
1 9 62) .
There are n o reports of ribose fermentation by
S. lactis .
N on-carb ohydrate c ompounds do not appear to be
fermented to any appreciable extent, with the exception
of arginine.
Arginine catabolism by S. lactis produce s
adenosine triphosphate ( ATP) and the enzyme s involve d h ave
been characteri ze d (Korzenovsky & We rkman, 1 9 5 3, 1 9 54) .
The literature c ontains no detailed reports on the
chemical composition or structure of S . lactis organisms
13 .
and there are no indications of the pre sence of any
' storage ' polymers , such as polysaccharide .
The industrial importance of S . lacti s has led
to several survival studies in milk cultures at low
temperatures (Gibson, Lande rkin & Morse 1 9 6 5 , 1 9 6 6 ;
Lamprech & Foste r , 1 9 6 3 ;
1966 ) .
Cowe l l , Koburge r & Weese ,
Howe ve r , the re are no reports of survival
studies at growth temp e rature s .
PART I I .
i)
SURVI VAL OF VEGETATI VE MICROORGANISMS
DEFINITION OF TERMS
Viability of a microbial population refers to the
proportion of the organisms capable of multiplication
when provide d with optima l growth conditions (Post gate ,
19 67 ) .
The refore , viability is norma lly assessed by
growth me a surements and gene rally the surviva l potential
of a population must be define d experimentally ( Dawe s &
Ribbons , 1 9 64) .
A dead organism may not nece ssarily
be metabolically ine rt.
For example , it h a s been
claime d that s ome b acteria retain functional osmotic
barriers a fter de ath (P ostgate & Hunter , 1 9 62) .
Some
authors have use d the terms a ge ing or senescence in
connection with studies on the activities of starving
microbial populations .
Howe ve r , the extrapolation of
the se concepts to unicellular microorganisms , in the
sense norma lly applied to higher organisms , has been
questioned by s ome workers (see Clifton , 1 9 6 6 ;
P o stgate ,
1967 ) .
ii)
METHODOLOG Y
Many inve stigations of microbial survival under
conditions of stre s s have been reporte d .
The se stre sse s
include de siccati on, freezing, heating, exposure to
adve rse pHs, osmotic pre ssure, r a diation, light and
14.
toxic materials .
It may be conside red that starving
popul ations are subjected to the Iprima ryl stre s s of
nutrient deficiency when they are incub ate d at normal
growth temperature s .
other
I
Starvation environments without
secondary I stresses maybe difficult, if not
impos sible, to achieve in practice (Po stgate, 1 9 6 7 ) .
Ideally, organisms should be remove d from a growth
me dium without centrifugal damage, washed and re­
suspende d in non-nutrient buffer solution a dj uste d to
The
the optima l survival pH and ionic concentration .
buffer shoul d contain stabili zing met a l i ons plus a
chelating a gent to remove any toxic metal i ons pre sent .
The re should also be minima l change in pH, buffe r
composition, aeration or temperature during this
process, since there is considerable e vidence that
starving organisms become hypersensitive to secondary
stre sses (P ostgate, 1 9 6 7 ) and many b acterial species
appe ar to de ve lop increased nutritional re quirements
under stress .
P ostgate & Hunter ( 1 9 62 ) demonstrate d
that buffer solutions prepared from the purest
available materials often containe d impuritie s at a
t oxic level .
Contaminant copper was i dentified and
its effect neutralized with ethylene diaminetetra-acetate
(EDTA) .
Garvie ( 1 9 5 5 ) found that E . coli multiplied
on contaminant nutrients in buffer solution prepare d
from specially purifie d salts dissolve d in distilled water.
Similarly, Sobek, Charba & Foust ( 1 9 6 6 ) reported growth
of ' starve d ' Azotobacter agilis in tris buffer suspensions .
Many worke rs have routine ly subjected organisms to low
tempe rature s or distille d water washes during ha rve sting
procedures (Burleigh & Dawe s, 1 9 6 7 ;
1965;
Clifton & Sobek, 1 9 6 1 ;
Dawes & Ribb ons,
McGrew & Mallette, 1 9 62 ) .
These procedure s are probab ly best a voided in view of
the conside rable sensitivity of many organisms to cold
shock and osmotic shock (Strange & Dark, 1 9 62 ;
& Hunter, 1 9 62 ) .
P ostgate
Howe ver, under certain conditions
15 .
water-washe d suspensions of Ae robacter aerogene s were more
resistant to sta rvation than bacteria washe d with saline­
phosphate buffer
( Tempe st
& Strange , 1 9 6 6) .
Apa rt from the findings of P ostgate , Strange and
collaborators ( see re view by P ostgate , 1 9 6 7), the literature
c ontains few precise measurements of bacterial survival
under es sentia lly ' stre s s-free' conditions .
Earlier
studies on microbial survival at growth tempe rature s in
buffer solutions may have been complicate d to an
appreciable extent by the secondary stre sses discussed
previously, difficulties in counting, problems of cell­
a ggregation and ' cryptic growth! .
Available methods for the assessment of viability
have been crit ically reviewe d recently by P o stgate ( 1 9 6 7) .
The only method which is both convenient and unamb iguous
appe ars to be the micro-slide culture method of P ostgate ,
Crumpton & Hunter ( 1 9 6 1) .
This procedure give s accurate
viabilities (% viable / t otal organisms ) in the range
5 - 1 0 0% viab ility by short term incubation of sample
populations on agar films fol lowe d by differential c ount­
ing of viable and de ad organisms us ing a phase c ontrast
microscope .
This method is most suitable for populations
of unice llular, aerob ic or facultative organisms with
uniform division lag time s .
Gross inaccuracies a rise
with organisms which tend to a ggre gate
( Burleigh
& Dawe s ,
1 9 67) o r with filamentous o r cha in-f orming organisms which
tend to fragment .
As clumping or fragmentation of
starving organisms seems quite pre valent , their effect
on viability data must be assessed in the inte rpretation
of re sults .
With populations having varying division lag
time s , care must be taken to avoid overgrowth of colonies.
Howe ve r , sufficient incubation time shoul d be a llowe d to
permit division of all survivors.
As we ll a s accurate
viable c ounts , accurate total counts a re also necessary
to ade quately de scribe survival cha racteristics of a
starving population since lysis of de ad organisms or
16.
' cryptic growth ' may obscure studies on the physiology of
sta rvation.
' Cryptic growth ' (Ryan, 1 9 5 9 ;
Harrison, 1 9 6 0) is
caused by nutrients le aking from dea d or lysed organisms
a llowing growth of survivors without a net increase in
bacte rial mass ;
may re sult.
an increase in t otal b acterial numbers
Thus a new population may be formed s o that
re sults cannot b e inte rpreted in terms of the origina l
populati on.
C ryptic growth is like ly t o be of
particular importance with organisms which can utilize
a wide range of substra tes for energy, e specially when
these organisms a re starved at high population densities
or in the presence of an a dde d energy s ource.
The
probable existence of cryptic growth in many published
experiments involving starved suspensions unde rgoing
' substrate-accelerated de ath ' , or in ' ma intenance ene rgy '
expe riments, may invalidate many of the c onclusions drawn
by s ome workers ( se e re view by Postga te, 1 9 6 7) .
The method of routine culture of an organism, the
growth rate, me dium composition and the growth phase at
which organisms a re harve ste d from batch culture, may have
pronounced effects on the survival of bacte ria ( Strange,
Dark & Ne ss, 1 9 6 1 ;
P ostgate & Hunte r, 1 9 6 2) .
Little
reliance can be placed on the survival mea surements of
workers who failed to take all the se f actors into account .
Herbert ( 1 9 6 1) and Neidhardt ( 1 9 6 3) re viewe d reports which
illustrate h ow gre atly the chemica l c omposition of micro­
organisms may vary in re sponse to change s in the c omposition
of the growth me dium .
Such complications in making survival mea surements
stre ss the nece ssity for care in de signing experiments in
studie s on the survival prope rties of mic roorganisms .
( iii )
(a)
17.
RESULTS O F VIABILITY MEASUREMENTS ON MICROORGANISMS
S TARVED AT THE GROWTH TEMPERATU RE UNDER ' MINIMUM­
S T RESS ' CONDITIONS
Effect of i onic environment on survival
Early work on the survival of starve d b acte r ia in
a queous suspensions has been discussed briefly by P ostgate
& Hunter ( 1 9 62) .
Although some of this work is of doubtful
significance because most of the compl ications mentioned in
the previous section were not appreciate d at the t ime , it
2+
was establishe d that the presence of the cations Mg
and
2
Ca t , and buffer solutions at certain pH values favoured
survival of coliform b acteria .
The work of P ostgate &
Hunter ( 1 9 62) confirme d these findings and established the
necessity for a dding traces of EDTA t o the buffer .
2+
. I
The a b so I ut e re qulremen
durlng
.
.
b act e rla
t f or Mg
growth (Webb , 1 94 8, 1 949, 1 9 5 1) is understandable from its
known functions as a co-factor in many enzymic reactions
and its role in the stabilization of membranes and
2+
r ib osomes .
As well as being essential for growth, Mg
decrease d the death rate of sta rve d organisms in buffered
suspensions ; the mechanism of this protective action has
n ot been clearly defined .
Tempest & Strange ( 1 9 6 6)
suggested that most of the intrace llul a r magnesium in
b acteria was associated with ribosomes and up to 2 5% of
the total magnesium may be loosely attache d to the cell
2+
surface .
Surface adsorbed Mg
was unaffected by
washing with distilled water but was desorbe d by washing
with sal ine-phosphate solutions ( Strange & Shon , 1 9 64).
Water-washed A . aerogenes organisms were more resistant
to starvation and other stresses than saline-phosphate
washe d bacteria ( Tempest & Strange , 1 9 6 6) and this has
2+
Thus
been attribute d to the retention of a dsorbed Mg .
2+
the import ant functional role that surface a dsorbed Mg
may have in bacteria should be considered when prepar ing
18 .
washed bacterial suspensions for studies of metabolic
a ctivity or surviva l .
Strange & Hunter ( 1 9 6 7 ) considered that the decreased
death rates of sta rve d A . aerogenes and E . coli in the
2+
presence 0f exogenous Mg
may h a ve b een due t 0: ( 1 ) A
stabil izing action on b a cterial ribosomes as shown by the
l ower rates of death and RNA degra dation during starvation
of strains of these organisms at growth and higher
2+
temperatures .
(Mg
is believe d t o be involved in at
least three aspe cts of ribosome structure and function:
(i ) conformation of the RNA strucutre , (ii ) association
of RNA with protein and (iii ) ribosomal aggregation
(see Rodge� 1 9 6 6 ) ) .
(2 ) An effect on b a cterial
metabolism indicated by the abolition of the lethal
effect of carbon energy sources on starve d b a cteria .
(3) A stabilizing effe ct on the permeability control
2+
mechanisms of bacteria which, in the absence of Mg ,
are susceptible to cold shock .
Although it has been
.
2+
shown that exogenous Mg de creases .the, death rate of
Gram-negative bacteria starve d at the growth temperature ,
2+
Burleigh &. Dawes ( 1 9 6 7 ) have found that a dded Mg
did
not affect the death rate of the Gram-positive organism
S arcina lutea , despite t he suppression of RNA
degradation.
Further evidence for the importance of the ionic
environment in bacterial survival is found in the
considerable body of evidence showing that EDTA
disorganizes the outer layer of bacterial cel l walls,
presumably by b inding or extracting certain cations
responsible for structural integrity ; the process
leading t o the rapid death of sensitive organisms ( see
Gray & Wilkinson , 1 9 6 5 ; Asbell & Eagon , 1 9 6 6 ) .
Other
ionic factors influencing the survival of microorganisms
were discussed by P ostgate ( 1 9 6 2 , 1 9 6 7 ) .
19 .
(b )
Effect of exogenous substrates on survival
P ostgate & Hunter ( 1 9 6 2 ) reported that cells of A . aerogenes
which ha d ceased growing because of glycerol exhausti on in the
me dium, die d more rapidly when washed and starved in phosphate
buffer cont�ining glycerol than in phosphate buffer alone .
This phenomenon was terme d ' substrate-accelerated death ' and
has since been investigated intensively for A . aerogenes (see
review by Postgate , 1 9 6 7 ) .
The phenomen on was demonstrated
with organisms from carbon- , nitrogen- and phosphate- l imited
�e dia but not with organisms grown under l imitations of
sulphate or magnesium ions ( P ostgate & Hunter, 1 9 6 4 ) .
A high
degree of substrate specificity was suggeste d and it was shown
2+
that the addition of e ithe r Mg
or the ' uncoupling ' agents
2 , 4-dinitrophenol and azi�, abolished glycerol-accelerated
death .
The increase d death rate was n ot accompanied by
accelerated breakdown of the osmotic barrier or cel l polymers
and surviving organisms showed long division lags which were
considered by P ostgate & Hunter ( 1 9 6 4 ) to indicate a slow
reclamation of lost cell material or the recove ry from
repression of enzyme synthesis .
Strange & Dark ( 1 9 6 5 ) repeated these experiments with
the same strain of A . aerogenes and found that substrate­
accelerate d death was less general than cla imed by P ostgate
& Hunter ( 1 9 6 4 ) and was in fact restricted to a n on-specific
lethal effect of carbon energy sources metabolized by
2+
However , Strange
A . aerogenes in the absence of added Mg .
& Hunter ( 1 9 6 6 ) later withdrew this conclusion having
succee de d in observing nitrogen-accelerated death .
The
discrepancies in these results from adj acent l ab oratories
arose from the appearance of a variant organism which
replaced the p arent strain of A . aerogenes after prolonge d
continuous �ulture (Strange & Hunter , 1 9 6 6 ) .
These
experiments emphasize the possible dangers arising from
mutation in the growth culture .
Strange & Dark ( 1 9 6 5 )
reporte d that the death rate increased with the rate of
20 .
2+
substrate metabolism while exogenous Mg
de creased the rate
of degradation of intrace llular RNA without significantly
affecting substrate metabolism .
The mechanism of substrate-accelerated death is not
fully understood .
However , Strange & Dark ( 1 9 6 5 ) attributed
glycerol-acce lerated death, at least in part, to the form­
ation of an unidentified toxic product of glycerol metabolism .
2+
It was suggested that a dde d Mg
prevented the accumulation
or formation of this product.
During glucose-accelerate d
death the bacterial ATP pool increased significantly whereas
2+
in the presence of glucose plus Mg
there was l ittle change
It was
(see discussion of paper by Strange & Hunter , 1 9 6 7 ) .
suggeste d in this discussion that if all the b a cterial
a denosine diphosphate (ADP ) was conve rted t o ATP, then
oxidati ve phosphorylation would be impeded and t oxic
hydrogen peroxide might accumulate .
In this situation ,
2+
could exert its prote ctive effe ct by increasing ATP
Mg
hydrolysis .
Such an explanation would be consistent with
the report that 2 , 4-dinitrophenol abolishe d glycerol­
accelerated death (P ostgate & Hunter , 1 9 6 4 ) .
However , any
proposed mechanism should be consistent with the fact that
while exogenous glucose accelerates death, the death rate
de creases in parallel with increasing intracel lula r polyglucose
leve ls (Strange et a l . , 1 9 6 1 ) .
P resent data does not establish whether accelerated
death by inorganic substrates, such as phosphate and ammonium­
ion , occurs by a mechanism similar to carb on substrate­
a ccelerated death but P ostgate & Hunter ( 1 9 6 4 ) have pointed
out that it is l ikely t o be intimately involved in the control
me chanisms governing the synthesis of constitutive material
in the cel l .
Although a considerable amount of data has
been publishe d on substrate-accelerate d death, it is n ot
clear whether this phenomenon occurs with organisms
harvested from the logarithmic growth phase in batch culture
where there are no l imiting nut rients .
No b a cterial lysis
21 .
or change in total cel l numbers occurred during substrate­
accelerated death and the good agreement between viability
results by plate count and slide culture (P ostgate & Hunte r ,
1 9 64 ) w as considered by these authors to preclude the
possibi lity of b iosynthesis taking p lace and giving rise
t o unbalance d growth ( see discussion of pape r by S trange
& Hunter , 1 9 6 7 ) .
Substrate-accelerate d death may have
some features in common with the ' suicida l ' behaviour of
certain microbial mutants ( e. g . thymine-less E. coli ,
Barner & Cohen, 1 9 5 6 ) which show accelerated death when
deprive d of an essential metabolite in an otherwise complete
growth medium .
In contrast to the effect of carbon substrates on
starving organisms discussed above , there are seve ral
recent reports of decrease d death rates resulting from the
repeated a ddition of smal l amounts of glucose to starving
populations of B. coli ( Clifton , 1 9 6 6 ; McGrew &
However , the
Mallette , 1 9 6 2 , 1 9 6 5 ; Mallette , 1 9 63 ) .
e valuati on of these results is difficult since the
e xperimenta l design did not preclude regrowth ( see
previous discussion ) .
Postgate & Hunter ( 1 9 6 2 ) found that the additi on of
amino acids and vitamins to starving populations had no
effect on survival and the protective effect of the basal
me dium, without a carbon source , was completely attribut�
able to its trace element content.
There appear to have
been no une quivocal reports of decreased b acterial death
rates in the presence of an energy source without
concomitant growth, although the exper iments of Tempest ,
Herbert & Phipps ( 1 9 6 7 ) involving growth in chemostats
at low dilution rates would indicate that such a situation
is possible.
(c)
Effect of bacterial density on survival
A bacterial density effect on survival was first
investigated by Harrison ( 1 9 60 ) .
Dense populations were
22 .
found to have l ower death rates than sparse populations
although an optimum density was claimed for survival above
which the de ath rate in crease d .
These findings were later
confirmed by Postgate & Hunter ( 1 9 63 ) who demonst rated that
the population density effe ct was not an experimental
artifact .
Howe ver , the ' re verse d ' death rate at high
bacterial concentrations reported by Harrison ( 1 9 60 ) , was
not observe d .
Although limited cryptic growth o ccurred
in Harrison ' s high density populations it was considered
that this could n ot have contribute d significantly to the
decrease in death rate .
The intrinsic factors involve d in the population
density effe ct have not been resolve d .
Harrison ( 1 9 60 )
suggeste d that starving organisms release substances which,
if they reach a critical concentration , may be taken up by
surviving o rganisms and permit them to maintain viability
for a longe r time .
It was concluded that the se crete d
material responsible provide d an energy source for
maintenance .
Starving Gram-negative organisms are known t o degrade
intracellula r RNA at rapid rates, espe cially in the absence
2+
of exogenous Mg , so that the magnesium associated with
rib osomes is release d .
This fact , together with the
observations that population density effects occur in
free zing damage ( Harrison , 1 9 5 5 ) , cold shock ( Strange &
Dark , 1 9 6 2 ) , thermal death ( Strange & Shon , 1 9 6 4 ) and
substrate-accelerated death ( P ostgate & Hunter , 1 9 6 4 ) ,
2+
is known to be protective , suggests
where exogenous Mg
that the se creted material responsible for the population
2+
However , P ostgate & Hunter
density effect may be Mg .
( 1 9 63 ) still observe d a population density effe ct in the
2+
presence of optimal exogenous Mg
and could not dete ct
2+
Mg
in the filtrates from d ying populations .
--- -
( iv )
23 ·
CATABOLISM AND T URNOVER OF CELL CONSTITUENT S IN
VIABLE , STARVED BACTERIA
The progressi ve loss of c ell constituents whi ch generally
oc curs when bacteri a are starved in buffer at the growth
t emperatur e , is no rmally a r esult of an imbalance in the total
anabo lic and catabolic reactions .
It may also r esult from
other processes such as the l eakage or secretion of cell
components and intracellular pools .
Limited r esynthesis
or turno ver of c ell polymers may occur but the net metabolism
is catabolic and must eventually result in d eath of the
organism ( Strange , 1 9 67 ) .
The overall metabolic a ctivities
of starved bact eria are normally r eferred ,to as their ' endogenous '
metabolism whi ch , a ccording to Powell ( 1 9 67 ) , is effecti vely a
measure of the rate at whi ch bacteri a breakdown thei r own mass .
This fi eld has b een extensively investigated with a erobi c
organisms and published work has b een revi ewed in a symposium
( L amanna , 1 9 6 3 ) and by Dawes & Ribbons ( 1 9 6 2 , 1 9 6 4 ) .
The
functions of endogenous metabolism , as envisaged by Dawes &
Ribbons � 9 6 2 ) , include the provision of energy : for turnover
of protein and nucleic a ci ds , osmotic r egulation , pH contro l
and the supply of suitabl e substrates for the r esynthesis of
Components reported to
essenti al bacterial constituents .
be d egraded in starved organisms include carbohydrat e , RNA ,
prot ein , fr ee amino a ci ds , p eptides , lipids and c ertain
specializ ed ' r eserve ' materials .
Cellular constituents which
have not b een implicated as substrat es for endogenous
metabolism include DNA , c ertain cell wall and membrane polymers
( Dawes & Ribbons , 1 9 6 4 ) . Th e rates and orders of substrate
degradation and the general patt ern of endogenous metabolism
vary ( 1 ) in different o rganisms , ( 2 ) with the growth
conditions and henc e the chemi cal and physiolo gical state of
the o rganism and ( 3 ) with the physi co�chemi c a l conditions
of the starvation environment ( Strange , 1 9 67 ) .
-
24 .
In certa in nutrient conditions, many bacteria accumulate
relatively l arge amounts of polymers whi ch have been considered
as specialized carbon and/ or energy-st ores, analogous t o the
lipid and glycogen rese rves in animals (see reviews by
Wilkinson , 1 9 5 9 ; Wilkinson & Duguid, 1 9 6 0 ; Duguid &
The principal compounds
Wilkinson , 1 9 6 1 ; Neidhardt , 1 9 63 ) .
which have been implicate d as carb on and energy reserves
in b a cteria are intrace llula r polysaccharides, in particular
glycoge n , and intracellular l ipids , especially polye;8 hydroxybutyrate (PHB ) .
T hese two polymers have n o other
known function in b a cteria (Wilkinson, 1 9 59 ) .
Capsular
and cel l wal l polysaccharides do not normally a ct as energy
stores a ccording t o Wilkinson ( 1 9 5 8 ) and Duguid & Wilkinson
( 1 9 6 1 ) . P olyphosphate has been considered to a ct as a
phosphorus and/ or energy reserve (see Duguid & Wilkinson,
1 9 6 1 ) , a lthough its energy reserve function in a strain of
A . aerogenes has been questione d (Harold & Sylvan , 1 9 63 ) .
Before a particular substance can be considered to
have a spe cial ized reserve function , certain criteria must be
met (Wilkinson , 1 9 5 9 ; Wilkinson & Munro , 1 9 6 7 ) .
These
authors considered that the reserve must a ccumulate in
conditions where the supply of the necessary components for
its synthesis is in excess of growth demands .
They a lso
suggeste d that the reserve must be metabolized when exogenous
substr ates can n o longe r support growth , yiel ding energy
and intermediates that can be utilized by the cell for
' ma intenance ' .
Wilkinson ( 1 9 5 9 ) considered it probable
that the glycogen accumulate d by E . coli (Holme & P a lmstierna ,
1 9 5 6 b ) and the P HB in Bacillus megaterium (Macrae & Wilkinson ,
1 9 5 8 ) fulfilled these criteria .
Accumulation and utilizat­
ion of the reserve in the natural environment of the o rganism
However , this criterion
was a lso considered essential .
cannot n ormally be established because of the difficulty in
reproducing the natura l environment of most b a cteria .
The growth conditions necessary for the accumulation
of glycogen in E . coli (Holme & P almstierna , 1 9 5 6a , b ; Ribbons
25 .
& Dawe s , 1 9 63 ) and of PHB in B . megate rium ( Macrae & Wilkinson ,
1958 ;
Wilkinson & Munro, 1 9 6 7 ) , have been extens ively
inve stigate d .
In e ithe r batch o r continuous culture systems ,
nitrogen-limiting growth conditions or carbon source exce s s ,
we re e ither e ssential o r stimulatory for the accumulation of
these re serve s .
Synthesis of these storage compounds can
therefore t ake place independent of growth .
As wel l as
de gra ding the i r re serve s on starvation , bacte ria may also
de gra de othe r cel l c onstituent s , s ometimes after the re se rve
is e xhauste d ( Dawe s & Ribbons , 1 9 6 5 ) .
Howe ve r , prefe rential
utilization of re serve s has only been reporte d for s ome spe c ie s .
Some bacterial spe cie s cannot accumulate re serve s but this doe s
n ot imply that the se organisms are incapable of endogenous
respiration ( see Campbe l l , Gronlund & Duncan , 1 9 63 ) . When
such bacteria a re starve d , the degra dation of ' ba sal ' materials
such a s RNA , protein and free amino acids may occur ( see Dawes ,
1967) .
At rapid growth rate s the nucleic acids of E . coli are
stable , whereas in starving o rgani sms only DNA is stable and
RNA unde rgoes c onside rable de gradation ( Mandel stam, 1 9 6 0 ) .
T he de gra dation of RNA appears to be wide spread in starve d
b a cteria and has been demonstrate d i n L . casei ( Holden, 1 9 5 8 ) ,
A . aerogene s ( Strange et al . , 1 9 6 1 ) , E . coli ( Dawe s & Ribbon s ,
1 9 6 5 ) , Sarcin a lutea ( Burleigh & Dawes , 1 9 6 7 ) and P se udomona s
aeruginosa ( Gronlun d & Campb e l l , 1 9 63 ) . The ribose portion of
the RNA was generally oxidize d and the nitrogen normally
appeared in the suspending buffer as ammonia and free base s .
Wade ( 1 9 6 1 ) demonstrate d two possible r oute s of enzymic
bre akdown of RNA depending on the pre sence or absence of
magne s ium .
The protein fract i on of
E.
coli i s also stable at
rapid growth r ates according to Mande l st am ( 1 9 60 ) but net
de gra dation may occur in starvation conditions ( Strange et al e
1 9 6 1 ; Postgate & Hunter , 1 9 6 2 ; . Dawes & Ribbons , 1 9 6 5 ) .
Loss
of prote in from all the ultra-centrifugal fractions of starve d
,
26.
A . aerogenes occurre d (Strange , Wade & Ness , 1 9 63 ) while
Strange ( 1 9 6 6 ) presented e vidence that a daptively forme d
� -gal actosidase in E . coli was degra de d at a higher rate
on starvation than the overall rate for cel l protein ; this
indicates that some proteins are less resistant to degra dat�
i on than others .
The products of protein catabolism which
appeared in the suspending buffer , were normally CO 2 and
ammoni a with only trace amounts of amino acids .
Peptone�
grown S . lutea degra de d RNA and intracellular free amino
a ci ds when starve d , while protein , polysaccharide and DNA
were stable in this situation ( Burleigh & Dawes, 1 9 6 7 ) .
The free amino acid and peptide pool in peptone­
grown S . lutea was considered by Dawes & Holms ( 1 9 5 8 ) to
constitute the main endogenous substrate .
The pool was
reduced to a l ow leve l on starvation and was n ot replenished
from cell protein .
Glutamate a ccounted for 2 0 % of the
free amino acid pool and sign ificant depletion of �he pool
could not b e attributed t o the secreti on or leakage of
amino acids .
Although polyglucose is a ccumulated by
glucose-peptone grown S . lutea organisms, this compound does
n ot exert the same sparing a ction on n it rogenous substrates
( Ribbons & Dawes , 1 9 63 ) as it does in starved E . coli (Dawes
& Ribbons , 1 9 6 5 ) .
Early reports by Stephenson & Whetham ( 1 9 2 2 ) indicated
that growing Mycoba cterium ple i accumulated l arge quantities
of lipid, especially in the presence of a cetate and it was
suggested that ' neutra l ' lipid was degra de d in starve d
organisms .
However , the relatively crude extraction ,
identification and assay te chniques make this data difficult
No reports of endogenous glyceride or fatty
to e va luate .
a cid catabo lism by starving bacteria have since been publishe d
a n d an energy storage functi on for triglyceride i n b a cteria
has not been establ ished ( see Robertson, 1 9 6 8 ) .
When b a cteria are starved of a nitrogen source , net
27 .
protein and RNA synthesis stops but protein and RNA turnover
may continue for several hours . Degradation of pre-existing
protein and RNA provides amino acids and bases for resynthesis
( see reviews by Mandelstam, 1 9 6 0 , 1 9 6 3 ) . During starvation
of E . coli at the growth temperature , degradation of protein
occurred at a rate of about 5%/h� for a least 4hr . This
was balanced by an equal rate of resynthesis resulting in no
net protein loss in 4h� (Mandelstam, 1 9 5 7 , 1 9 5 8 ) . Bacterial
lysis , as measured by the release of � Mgalactosidase into the
suspending buffer, was not appreciable in these experiments
suggesting that only intracellular protein turnover occurred .
Subsequently, Levine ( 1 9 6 5 ) demonstrated that intercellular
protein turnover occurred at a lower rate of 0 . 1 6-0 . 1 8%/hn in
starved E . coli . Not all the proteins of nitrogen-starved
E . coli are subj ect to degradation at equal rates (Willetts ,
1 9 67 ) . Ribosomal protein and RNA degradation occurred at
similar rates (Mandelstam & Halvorson, 1 9 60 ) and Mandelstam
( 1 9 6 3 ) considered it probable that the starvation conditions
which caused protein breakdown also caused RNA breakdown .
Protein turnover was not measured over extended starvation
periods by Mandelstam, but Schlessinger & Ben-Hamida ( 19 66 )
reported declining, although significant protein turnover
in nitrogen-starved E . coli for at least 20hr . Ben�Hamida
& Schlessinger ( 1 9 6 6 ) recorded a much lower rate for RNA
turnover in nitrogen-starved E . coli . These authors concluded
that ribosomes are not resynthesized and that the net effect
of the turnover process was to transfer amino acids and
nucleotides from a metabolically useless excess of ribosomes
to the soluble proteins, energy supply and . reserves required
for subsequent adaptation .
The protein turnover rates ( 5%/hr.) reported by
Mandelstam ( 1 9 57 , 1 9 5 8 ) were considerably greater than those
measured by Dawes & Ribbons ( 1 9 6 5 ) in starved E. coli ( 0 . 6%/h�) .
Dawes & Ribbons ( 1 9 6 5 ) suggested that the difference may
rerlect a greater capacity for turnover in Mandelstam ' s
28 .
me dium whi ch was complete except for a nitrogen s ource , as
oppose d to starvation in the ir simple s alt me dium .
Protein
turnover occurred in starve d glucose-grown E . coli conta ining
glycogen but when all of the ce llular glycogen had been use d ,
ammonia wa s released which w a s considered by Dawe s & Ribb ons
to indicate net prote in de gradation .
( 1965)
Tryptone -grown
E . coli was de void of glycogen and net protein degra dation
occurred from the be ginning of the starvation period
( Ribbons & Dawe s ,
1 9 6 3.) .
The se findings may be compared
with the ob servations of Duncan & Campbell
( 1962 ) ,
who f ound
that starve d P . ae ruginosa re leased ammonia which was
reincorporated on a ddition of exogenous glucose .
Clifton
( 1966)
S imilarly,
reported reincorporation of ammonia by starve d
E . coli when gluco se was adde d and the highe r viabilities
with a dde d glucose may indicate that re synthe sis favours
surviva l .
It i s now clear that while protein degra dation occurs
at similar rates in b oth nitrogen- and carbon-limited me dia
(Willett s ,
1967 ) ,
the rate of prote in re synthe sis is ve ry
dependent on the pre sence of a carb on s ource ( Schle s s inge r
& Ben-Hamida ,
(v)
1 9 66 ) .
THE ROLE OF ENDOGENOUS METABOLISM IN THE SURVIVAL OF
STARVED BACTERIA
Many of the published pape rs on the endogenous
metabolism of bacteria do not include viability me a surements
so that corre lations with survival are not possible .
Ba cteria starve d in buffer at the ir optimum growth
temperature may survive for hours or days but prolonge d
starvation ultimately leads to death ( Strange ,
1 9 67 ) .
The
reasons for b a cterial death in starvation environments are
probably impossible t o define but s ome authors have cla ime d
that certain inte rpretations can be exclude d .
For instance ,
starve d A . aerogenes was cons ide red to maintain functional
memb ranes a fter death ( P o stgate & Hunte r , 1 9 6 2 ) .
McGrew & Ma llette
29 .
( 19 6 2 )
claime d that the continuous
a ddition of sma ll amounts of glucose to sta rve d E . coli
sustaine d viability for s e veral days without concurrent incre ase
in cell mass o r numbers .
They reported that extrapolation of
the curve of turbidity increment versus concentration of
exogenous ene rgy source to zero growth , gave a reproducible
interce pt at a definite positive level of ene rgy s ource .
This finding provide d e vidence for an ' energy of ma intenance '
for the survival of starving bacteria .
The se and othe r
attempts to demonstrate a n d me a sure the b a cterial maintenance
re quirement (Mallette ,
re view by Dawe s &
196 3 ; Marr, Nilson & Clark 1 9 6 3 ;
Ribbons , 1 9 6 4 ) have since been wi dely
see
criticized on the grounds that the se re sults could be
interprete d in terms of cryptic growth ( se e Dawe s & Ribbons ,
1964;
Clifton ,
1966 ;
P ostgate ,
Howe ve r , if cell
1 9 67 ) .
constituents are turning over and the membranes remain
functiona l , then energy is require d ( see �awe s & Ribbons ,
1964) .
While the pre sence of a suitable exogen ous energy
s ource may susta in viability for a limited period without
growth ( Clift on ,
1966 ) ,
the re a re no reports of starving,
vegetative cells which survive indef initely .
P ostgate & Hunte r
( 1962)
grew A . aerogenes in a
chemostat at decre a s ing dilution rate s until de a d o�ganisms
ma de a contribution to the ste ady state p opulation .
Over . this
range the re wa s a p rogre ssive increase in doubling t ime and
de cre ase in b a cte rial yield as measure d by mas s increase /
glycerol oxidized ( se e Tempest et a l . ,
1 9 67 ) .
This re sult
may be interprete d in te rms of a maintenance re quirement .
With carb on-, sulphate- or magne sium-limne d chemostat culture s ,
Postgate & Hunte r
( 1962)
were unable t o provide A . aerogene s
organisms with j ust sufficient nutrient to maintain themselve s
indef initely without divi ding and they conclude d that the se
b a cteria were ob lige d to multiply or die .
Essentially
similar re sults we re obta ine d by Tempe st et a l . ,
nitrogen-limite d A . aerogenes .
( 19 6 7 )
with
30 .
It has been sugge ste d that the metabolism of endogenous
substrates by starving b a cteria is ne cessary for their survi va l ,
s o that b a cteria which conta in large amounts of ' storage
polymers ' may _ have an a dvantage for survival ( see Lamanna &
Ma llette , 1 9 5 6 ;
Duguid & Wilkinson, 1 9 6 1 ) .
Although ene rgy
yielding reactions may occur in starve d bacteria and are
essential for surviva l , the amount of ene rgy required for
sur vival has not been e stablishe d .
For many microorganisms ,
indications a re that if an energy s ource is a va ilable , then
it is metab olized at a rate in exces s of that whi ch might be
expected for maintenance re quirements .
This initial rapid
utili zation of rese rves by s ome bacteria sugge sts that the y
may not ha ve a maintenance role ( see Dawe s & Ribbons , 1 9 6 4 ;
Strange , 1 9 6 7 ) .
The glycogen in glucose-gr own E. coli , whi ch
amounte d t o as much a s 2 3 % of the ce llular dry we ight , was
completely oxidized in 1 -3h� yet viability remaine d unchange d
for the f irst 1 2h� of starvation ( Dawe s & Ribbons , 1 9 6 3 ,
1965) .
A corre lation between the pre sence of a reserve
material and enhanced capa city- _ for survival has only been
found with s ome of the organisms studie d .
Glycogen-rich
A . aerogenes survive bette r than the corre sponding glycogen
def icient organisms ( Strange et a l e 1 9 6 1 ) , whereas glycogen­
rich S . lutea die more rapidly ( Burleigh & Dawe s , 1 9 6 7 ) .
Glycogen metabolism suppre ssed the release of ammonia from
starve d E . coli and A . aerogenes but not from starved S . lute a .
Dawe s & Ribb ons ( 1 9 6 3 ) sugge ste d that possession of glycogen
was not an a id in the survival of E . coli ( see re view by Dawes
& Ribb ons , 1 9 6 4 ) .
Survival of Micrococcus hal odenitrificans
and A . agilis appe a r s to b e related t o the initial P HB content
of the cells ( Sie rra & Gibb on s , 1 9 6 2 ;
Sobek et a l . , 1 9 6 6 ) .
In starved o rganisms with high PHB leve l s , the endogenous
re spiration rate and viability remaine d unalte red for 9 6h�
( Sierra & Gibb ons , 1 9 6 2 ) ;
the const ant rate of de gra dation
perhaps indicating s ome de gree of efficiency .
When the PHB
31.
level fell to a critical va lue , the respiration rate and
viability fell rapidly .
Similar organisms with low initial
P HB content showe d a rapid de cline in respiration rate and
viability from the onset of starvation .
The presence of PHB
was conside r e d to exert a sparing effect on cell prote in
( Sobek et a l . , 1 9 6 6 ) .
It woul d appear that organisms with
low respiration rates may be better e quippe d for surviva l .
Howe ve r , the re is no e vidence to sugge st that ve getative
bacteria a re capable of adapting the ir metabolism to
starvation con ditions .
P rofound change s in the composition of starved
b a cteria without concurrent viability loss are well
documente d .
Up t o 2 5% of the total cell prote in was
catabolized bef ore significant de ath of A . aerogene s o ccurre d
( Strange et a l . , 1 9 6 1 ) .
It appe ars that RNA is to some
extent expendable in that up to 50% of the ribosomal RNA of
s ome organisms can be metaboli ze d without de ath taking
place .
Within a single spe cie s , fast growing organisms
conta in much more RNA ( S chaetchter , Maa l oe & Kj eldgaard,
1958;
He rbert, 1 9 5 8 ;
Tempe st et a l . , 1 9 6 7 ) and die more
slowly than s l ow growing organisms ( P ostgate & Hunter ,
1962 ) .
Although many conditions whi ch de lay RNA degradat­
2+
( Strange & Hunter , 1 9 6 7 )
ion , such as the pre sence of Mg
o r D 0 ( Lovett , 1 9 6 4 ) , also de lay de ath , n o absolute
2
corre lation .between RNA de gra dation and de ath rate has been
e stablishe d ( se e P o stgate & Hunter , 1 9 6 3 ;
Burleigh & Dawe s ,
1967 ) .
When the DNA of E . coli was spe cifically labe lle d
with tritium, the organisms died more rapidly a fter a period
of cold storage than organisms s imilarly labelled in the ir
RNA and protein ( Ra chme ler & P ar de e , 1 9 6 3 ) .
The loss of
viability was attributed to r a diation dama ge t o the DNA,
indicating that inta ct DNA is required for b a cterial
viability and en zyme synthe sis .
32 .
Endogenous metabolism results in ATP generation but
a correlation between the ATP c ontent of starve d A . aerogenes
organisms and their survival properties could not be shown
( Strange , Wade & Dark, 1 9 6 3 ) . It was sugge ste d that su rvival
may be influenced by the total amount of AMP , ADP and ATP
present in the organisms , and a l s o by the ir ability to form
ATP when re quired .
Strange ( 1 9 6 1 ) conclude d that the loss
in ability of starve d A . aerogenes to synthesize � -ga l a ctosidase
was due to shortage of intermediates such as amino a ci ds , and
not ATP . It i s possible to define ene rgy requiring proce sses
in starving bacteria ( protein and RNA turnover , maintenance
of concentration gra dients a cross membranes , pH control , etc . , )
but the control and e conomy of these me chanisms a re i ll-defined
( see
Dawes & Ribbons , 1 9 6 2 ,
1964;
Strange , 1 9 6 7 ) .
In the past ( see Lamanna & Mal lette , 1 9 5 6 ) , it was
thought that respirati on was necessary to susta in viability of
aerobe s .
Howeve r , it is n ow wel l known that while many
starving organisms e xhibit high initial 0 2 - uptakes , the
0 - uptake may fall to a negligible leve l where significant
2
de cline in viability may not occur for a considerable period
( see Burleigh & Dawes , 1 9 6 7 ) . This sugge sts that the amount
of energy re quired for survival may be very small .
Glycerol dehydrogenase activity and the respiration
rate with glyce rol a s substrate , de cline d in parallel with the
viability of starve d A . aerogenes which were grown in a
glycerol-limited chemostat ( Postgate & Hunter , 1 9 6 2 ) . Burleigh
& Dawes ( 1 9 6 7 ) concluded that the survival of peptone-grown
S . lutea in aerobic starvation conditions could be correlated
with the ability to oxidize exogenous glucose and L-glutamate o
The se results suggest that the activity of the enzymes
catabolizing the energy s ource may be critical for surviva l .
Burleigh & Dawes ( 1 9 6 7 ) sugge sted that death of S . lutea was
probably caused by relatively non-specific inactivation of
enzymes , although it was not possible to attribute de ath to the
de gra dation or ina ctivation of any s ingle cel l constituent .
33.
Turnove r may represent a me chanism whereby cell
constituents , whose loss i s particularly l ike ly to result in
death, may be sele ctively reformed from dispensible material .
Schaechte r ( 1 9 6 1 ) removed organisms at interva l s from a
synchronized growth culture of Salmonel l a typhimurium and
transferred them after washing, to a non-nutrient phosphate
buffer .
Each group of organisms proce e de d to divide in the
buffer at the s ame t ime as they would have divide d in the
growth medium .
No b a cterial mass increase occurred , so
presumably extensive turnover was involve d - ( see also Dean,
1967) .
In experiments on the s l ow growth of A . aerogenes
in a chemostat , attempts to demonstrate what Postgate &
Hunte r ( 1 9 6 2 ) termed ' inheritance of l onge vity ' were
unsuccessful .
Howeve r , Harrison & La,wrence ( 1 9 6 3 )
demonstrate d that ' st arvation-resistant ' mutants could be
obtained from batch culture populations of A . aerogenes .
It would appear that the outstanding difference between thes e
mutants a n d the wild type was a slower e xponential growth
rate .
From a consi de ration of the growth rates of glycerol­
l imited A. aerogenes at de creasing dilution rates , Tempest
et al e ( 1 9 6 7 ) concluded that there i s a finite , temperature
dependent minimum growth rate for b a cteria .
As the dilution
rate was decreased, the doubling time tended t o a maximum
and the ' steady state ' culture viability progressively
diminishe d .
In natural environments such as seas, lake s ,
rivers and s o i l , the concentration o f substrates i s generally
The viable b a cteria in
low and possibly nitrogen-limiting .
such environments must be able to grow at very l ow generation
rates and it seems possible that the abil ity to grow very
slowly fa cilitates survival ( see Bls den , 1 9 6 7 ) .
Other factors
favouring survival in natura l environments probably include the
ability to form spore s , metabolic versatility and the ability
to withstand such stresses as temperature shock and desiccation .
In these habitats , organisms may be subj e cted to greater stres s
34 .
than in a constant experimental environment but despite these
stre s s e s , many organisms must survive f or very l ong periods .
Whereas experimenta l environments probably pe rmit maximum rate s
of metabolic a ctivity ( i . e . organisms a t the optimum growth
temperature in a que ous buffer ) and hence maximum rates of
breakdown and death , such f a ctors as l ower temperature s or
partial desiccation may re strict metabolic a ct ivity and
possibly death rate s , in natura l habit ats .
The faster A . aerogenes organi sms grow, the s lowe r
they die ( Postgate & Hunter , 1 9 6 2 ) .
Maximum growth rates
produce organisms of maximum s i ze and RNA content and the
greater ability of such organisms to maint a in viability may
be a result of an excess of s ome components in the organism .
Then the levels ? f components e ssential for viability may
have further to fall before death .
Organi sms growing at the
minimum rate for complete viability are c omparatively smal l ,
they have gre at ly reduced RN A and DNA levels and they die
very rapidly on complete starvation ( Postgate & Hunter, 1 9 6 2 ;
The se organisms appea r to be in a
Tempest et a l . , 1 9 6 7 ) .
state of ' minimum subsistence ' near death, s o that any
breakdown of essential cel l components is lethal .
When
placed in a rich growth medium, a consi de rable proportion of
these organisms die in the division lag phase ( P ostgate &
Hunter, 1 9 6 2 ) .
It has been shown that A . aerogenes organisms cannot
be maintained indefinitely in a non-proliferating, viable ,
steady state ( P ostgate & Hunter, 1 9 6 2 ; Tempest et a l . , 1 9 6 7 ) .
Starving bacteria can be considered to have a negative mas s
growth rate while endogenous metabolism continues ( P owell ,
1967 ) .
For a particular organism, this rate and the death
rate depend on such factors as the growth me dium composition
( hence cell compOSition ) , the growth rate which produced
the organism and the properties of the starvation environment .
35 .
AIMS OF THE PRESENT INVESTIGATI ON
Maintenance of bacterial cultures with hi gh acid
producing activiti es i s an important function of research
laboratori es s erving the dairy industry .
An understanding
of the factors affecting the survival and activity of cheese
' starter ' organisms i s therefore necessary and could also b e
important in evaluating the r o l e of ' starter ' organisms i n
chees e flavour .
Only a smal l number of reports on the metabolism of
starved lactic aci d bact eri a have b een published , possibly
b ecause oxygen consumption is not no rmally appreciable with
the s e organisms .
No correlations b etween viability and
endogenous metabolism have b een reported .
Most of the
reported studies on the metabolism of viable , starved bacteria
have involved organisms whi ch can accumulate ' reserves ' such
as glycogen or PHB .
Lacti c streptococci have no reported
' reserves ' and very limited catabolic activiti es , it was
considered that extension of · mdogenous metabolism studi es to
Streptococcus lactis could b e informative .
In addition , the
exacting growth requirements of S . lactis should a llow for
uncomplicated survival studi es in environments which would
l ea d to cryptic growth o f other o rgani sms .
Most reports on the endogenous metabolism and survival
of bacteria have b een concerned with Gram-negative o rganisms .
+
The report that exogenous Mg 2 suppressed RNA degradation but
had no effect on the survival of starved Sarcina lutea ( Burl eigh
& Dawes , 1 9 6 7 ) , ha s suggested that some of the important
survival cha racteristi cs of thi s Gram-positive organism may b e
markedly diff erent f rom Gram-negative organi sms .
Simi lar
findings were made by Dawes with two other Gram-positive
organisms ( s ee discussion , Strange & Hunter , 1 9 6 7 ) .
Further
investigations with Gram-positive organi sms have been indicated
to define mor e c l early the general pa rameters for bacterial
survi va l .
36 .
Although mi lk i s the normal culture medium for
S . lacti s , studi es on the physiology o f bacterial survival
r equi r e d efined environments and highly reproducible bacterial
cultures .
Whil e continuous culture propagation could b e the
b est growth procedure , the appropriate equipment and techni ques
wer e not available for the present . investigation .
Accordingly ,
preliminary experiments were undertaken to achieve controll ed
growth o f S . lacti s in batch culture .
Studi es on the
metabolism of viable , sta rved o rganisms a r e likely to b e
significant only if factors affecting survival are investigated
concurr ently .
' Minimum-stres s ' conditions must be defined
for the o rganism under �udy since it is not possible to
accurately extrapolate f rom dita obtained with other organisms .
Within the framework o f these defined conditions , further
investigations were undertaken to study the chemi cal changes
which occurred in starved S . lactis o rganisms with special
reference to changes in viability .
37.
METHODS
MICROBIOLOGICAL PROCEDURES
Organism .
Str ept oc occus l actis ML 3 , us ed in the pres ent
investigation , was obt ained from the c ollection of cheese
l st arter t organisms of the New Z eal and Dairy Res earch Institute .
Strain ML was selected bec aus e its short chain morphol ogy
3
fac ilitat ed the measurement of more accurate viability dat a for
reasons already discussed .
Identific ation .
The dat a used to check fue identity of S . l actis
ML periodic ally was obtained using the methods described by
3
The reactions
Robertson ( 1 9 6 1 ) and is summarized in T able 2 .
obs erved were similar to those report ed for S . l actis .
All
°
temper atures are given in C .
Culture maint enanc e .
Sinc e the method of propagation of lactic
streptoc occi has a profound effect on the propert ies of the
organisms , a det ai l ed proc edure for the maintenanc e of S . !actis
ML is outlined in Table 3 .
This procedure gave more r eproduc­
3
ible cultures than were obtained by maint aining the organism on
agar slopes at 0_4 0 with subculture at i ntervals of up to one
month .
Daily subculture subj ects the organisms to int ermittent
st arvation when growth c e ases .
However , in this environment
c ompl ete vi abil ity was maint ained until the next transfer and
the appearanc e of I st arvation-resist ant , mut ants on prol onged
subculture c ould not be demonstrated .
Innocul a were normally 1 % exc ept when initially transf erring
from skim milk to the routine medium when a 5% innoculum was
required for c onsist ent growth .
The reasons for the nec essity
of a l arge innoculum are not clear .
Reiter & Oram ( 1 9 6 2 ) found
that s ome S . l actis strains would not grow when transferred
38
T able 2 .
.
I dentification of Streptococcus l actis ML 3 •
T e st
Pre sent
study
Ce ll shape
ovoid cocci
Cell arrangement
Gram staining
Catalase
diplococci
Growth at
It
II
Growth in
10 0
4 50
4 . 0%
6 . 5%
+
It
cocci
ovoid cocci
( 1 9 57 )
+
+
+
+
( 1 50 )
+
-
...
+
+
?
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
nm
N
nm
N
+
NaC I
+
+
=
positive re action
+
=
-
=
negative re action
?
=
nm
=
n ot mea sure d
Bre e d et al e
( 1961)
-
NaCI
NH 3 from arginine
CO 2 from tyrosine
CO 2 from citrate
Growth at pH 9 . 0
Acid from : Gluco se
lactose
maltose
dextrin
Serological group
II
Robertson
variable rea ction
re action ill-define d
39 0
T able 3 .
Proc edure for maint enanc e of streptococ cus l actis ML •
3
S . Laptis ML 3
1
- maint ained in skim milk as desc ribed by
Whit ehead et al e ( 1 9 5 6 ) for S . c remoris HP ( DRI )
FREEZE-DRIED
STOCK CULTURE
- stored under vacuum at 4 °
Skim milk
1,
- subcultured daily for 2 da�s ( 3 0 ° )
Routine medium
- subcultur ed daily for 2 0 days ( 3 0 ° )
t
t
DEEP-FROZEN
STOCK CULTURE
( thaw 3 0 ° )
- cultur es frozen in s olid C O 2 + ether or
liquid N 2 , stored at - 7 5 ° to _ 8 0 °
Routine medium
- subcultured daily for 3 0 days ( 3 0 ° )
!
!
Experiment al syst ems
Note :
the deep-frozen stock cultures provided the regul ar
s ourc e of organisms , this stock was renewed from the freeze­
dried stock culture every six months .
40 .
directly from skim mi lk to a synthetic medium and requir ed an
It is wel l
int ermediate subculture in a broth medium .
establi shed that r epeat ed subculture of lacti c str eptococci
in skim mi lk o ver long periods may result in the development
of a ' l ess a ctive ' culture (Whitehea d , Briggs , Garvi e &
N ewland , 1 9 5 6 ) .
Since it i s not understoo d why this o ccurs
it was decided to limit th� period o f subcultivation according
to T able 3 .
Cultures deep -fro zen for six months were 5 0 - 8 0 %
viable o n thawing .
Culture media .
Syntheti c media for the growth o f S . l a ctis .
wer e r eported by Niven ( 1 9 44 ) , Anderson & Elliker ( 1 9 5 3 ) and
Reiter & Oram ( 1 9 6 2 ) .
Attempts to grow S . lacti s ML in each
3
of these media proved unsucc ess�ul ih the present study .
Reiter & Moller-Madsen ( 19 6 3 ) concluded that vitamins o r
amino a cids may b e essential , stimulatory or not r equired ,
depending on the extent of deficienci es o r imbalances in
the basal medium .
Growth antagoni sms varied for different
strains of the same organism and antagoni sms b etween structurally
related amino a cids could b e overcome by the use of p epti des .
Rather than r einvestigate the nutritional requi rements of
S . lactis ML 3 , casamino acids were used to r eplac e the
syntheti c amino acid mixture .
Even so , peptone was r equired
The culture medium finally
for maximum growth rates .
adopted contained ( g . /l . ) : l acto s e monohydrat e , 5 . 0 ;
sodium acetat e , 1 . 0 ; glyc ero l , 1 . 0 ; casamino a cids ( Di f co ) ,
5 . 0 ; peptone ( Difco ) , 0 . 5 ; L-asparagine , 0 . 1 ;
DL -tryptophan , 0 . 0 5 ; L-arginine , 0 . 1 ; adenine , 0 . 00 5 ;
guanine , 0 . 00 1 ; uraci l , 0 . 00 5 ; xanthin e , 0 . 00 5 ;
pyri doxine hydrochloride , _ 0 . 001 ; pyridoxal hydrochlori de ,
0 . 0002 ; nicotini c aci d , 0 . 001 ; calcium pantothenat e ,
0 . 001 ; biotin , 0 . 0001 ; riboflavi n , 0 . 0001 ; thiamin e
hydro chloride , 0 . 0001 ; f o l i c aci d , 0 . 0001 ; Q-aminoben zoic
aci d , 0 . 0002 ; NaHC0 3 , 4 . 2 ; Na HP0 4 , 4 . 2 5 ; KH P0 4 , 2 . 7 ;
2
2
MgS0 · 7H 2 0 , 0 . 1 ; F eS0 4 . 7H 0 , 0 . 001 ; MnS0 . 4H 0 , 0 . 001 ;
4
2
4
2
disodium ethyl enediaminetetra -a cetat e ( EDTA ) , 0 . 00 4 .
The
41.
l actose , NaHC0 3 and vitamin components we re sterili z e d
separately using a Se itz filte r , which h a d been pre -washe d
with 2 0 0ml . de-ion i ze d water , and a dded a septically t o the
other components which had been autoclave d at 1 1 5 0 for 2 0
min .
Such a me dium
The me dium had a final pH of 7 . 3 .
is not complete ly defined because of the pre sence of
casamino acids and peptone , in subse quent descriptions
it will be referred to a s the routine me dium .
The broth me dium which was use d occasionally, containe d :
l actose , 0 . 5% , Casamino acids ( Difco ) , 0 . 5% ; peptone ( Difco ) ,
0 . 1% ; yeast extract ( Difco ) , 0 . 2 5% ; MgS0 4 , 0 . 0 1% ; 2 �
M-EDTA;
Na 2 HP0 4 -KH 2 P0 4 buffer , 0 . 0 7 5M , pH 7 . 3 .
Growth conditions and resuspension procedures . All growth
cultures and washe d suspensions were incubated in a water
bath at 3 0 0 in static conditions unless otherwise state d .
o'r ganisms were prepared for s urvival studies a s follows .
After 1 0-20 daily transfers ( 70 - 1 4 0 generations ) in the
routine medium, growth phase organisms were inocul ated into
2 5ml. me dium and growth was followe d turbidimetrically .
At
the end of the growth phase , a sample ( usua l ly 6ml.) was
remove d and after centrifugation ( 3 0 , 0 0 0g/ 1 min.) , the pellet
of organisms was rinsed thoroughly and then dispersed in
sterile phosphate buffer ( see below ) . Following further
centrifugation ( 3 0 , 0 0 0g/ 1 min.) , the organisms were rin se d and
f inally suspende d in 5ml. sterile phosphate buffer ( pH 7 . 0 ) .
Small volumes ( 0 . 2ml.) of the washe d suspension were use d to
inoculate 1 0ml. phosphate buffer ( plus substrate s e tc . ,
were spe cifie d ) in metal-capped test tubes which were
Suspens ions prepared in
incubated in a water bath at 3 0 0 •
this manner conta ine d the e quivalent of 20 ± 2 )A' g. dry wt .
organisms/mI. ( e quivalent to about 6 x 1 0 7 chains/mI. or
8
1 . 6 x 10 cocci/mlJ and had an initial viability of more
than 9 9 % .
Usua l ly samples were remove d a t interval s and
42 .
placed dire ctly on agar slide s for viability measurements .
In experiments with dense populations s ome dilut ion was
ne cessary before doing viability determination s .
Where
the effect of a toxic metal or growth inhibitor was be ing
examine d the organisms were centrifuge d and washe d in
phosphate buffer before the viability determinat ion .
The phosphate buffer use d containe d Na 2 HP0 4 + KH 2 P0 4
( 0 . 0 7 5M-P0 4 , pH 7 . 0 ) plus 1 0� M-EDTA, unless otherwise
stated .
No me asurable change in pH occurre d with suspens ions
e quivalent to 1 0m� dry wt . organisms/mI .
All washing and
resuspension of cel l s in buffers was carrie d out with
a septic precautions at 3 0 0 •
Change s in temperature and
chemical environment were thus minimized and manipulation s
were completed i n about 1 5 min .
These proce dure s left
insignificant trace s of growth me dium ( e quivalent to a
1 0 - 7 dilution ) and. repeate d centrifugation had no measurable
A vortexe ffect on the survival curve s of the organisms .
mixer was use d for all mixing and resuspension operations .
Similar proce dure s were adopted for experiments on the
metabolism of starve d S . lactis organisms ( see experimenta l
results for details ) .
Some settling of organi sms occurred
after prolonge d starvation ; in the se experiments gentle
agitation was provide d by a magnetic stirrer and corre ction
was made for e vaporation loss by a dding the appropriate
amount of sterile water .
Samples of supernatant buffer
solutions we re obtaine d for chemical analyses by centrifugation
and filtration through a membrane filter ( 0 . 4 � ; Millipore
Filter Corp . , Be dford, U . S . A . ) . When analyse s on the bacteria
were re quire d, the packe d organisms were washe d in phosphate
buffer and normal ly resuspende d in wate r .
Bacterial mass
determinations were carrie d out at the s ampl ing t ime , while
suspension samples and ce ll-free supernatant solut ions we re
o
store d at -20 when not analyse d imme diately .
Growth measurement and cel l counting proce dure s .
Growth
43 .
was followe d by taking samples at intervals from the culture
and measuring the turbidity at 6 0 0 � with either a Be ckman DB
or a Zeiss PMQII spectrophotometer .
Dilutions we re made in
distille d water where neces sary, e quivalent dilutions in
phosphate buffer gave the same turbidities .
A correlation
between bacte rial dry weights and the turbidities of suspensions
was obtaine d by c a libration with suspensions of known dry
wt . /ml .
Fixe d volumes of suspensions in distille d water or
phosphate buffer were drie d to constant we ight at 1 0 0 0 ,
appropriate correction being made for the amount of solids
present in the buffer .
The proportionality between bacterial
mass and turbidity was examined with a range of starvation
environments after varying periods of incubation and was
found to be constant in all cases .
All cell masses are
recorde d as mass units dry wt . bacteria/mI .
Total counts were made in a Petroff-Hausser chamber with
a phase contrast microscope (magnification x 3 1 0 ) . The pre­
cautions recommended by Norris & Powe ll ( 1 9 6 1 ) were taken
except that there was no instrument available to standardize
chamber depth .
The four large squares at each corner of the
grid were counte d for each sample ( 40 0 - 5 0 0 chains each) .
Counts are of individual chains separate d by at least two
coccal diameters unless otherwise spe cifie d .
The number of
cocci/ chain was determine d by counting the individual cocci
in a total of 2 5 0 chains for each sample (magnification x 1 1 0 0 ) .
These determinations were made on buffer suspensions using a
phase contrast microscope with oil immersion and a glass
coverslip .
The criterion used for establishing when cocci
had divide d was , of nece ssity, subjective .
Cell division
was considered to be complete d when the outline of the
individual cocci in the chain re sembled touching sphe re s .
The estimate of the cocci/chain given by this method is
probably subject to a systematic error giving slightly lower
ratios ( see Schae chte r , Williamson , Hood & Koch, 1 9 6 2 ) .
44 .
Viabil ity measurement .
The pe rcentage of viable organisms
in re suspended systems wa s determine d by the slide-culture
The only modification to
method of Postgate et al e ( 1 9 6 1 ) .
the method found ne cessary was the use of stainle ss steel
annuli instead of brass annuli, to avoid copper toxicity .
The agar me dium use d had the following composition ( g . / 1 0 0ml . ) ;
lactose monohydrate , 2 . 0 ; Casamino acids ( Difco ) , 1 . 0 ;
peptone ( Difco ) , 0 . 8 ; yeast extract ( Difco ) , 0 . 1 ; agar
( Davis ) , 1 . 0 ; Na 2 HP0 4 , 0 . 2 5 ; MgS0 4 . 7H 2 0 , 0 . 0 5 ; final
The agar was filtere d (Whatman no . 1 5 ) before
pH 7 . 0 .
autoclaving and centrifuged hot before slide preparation to
remove debris .
Slide-culture s were incubate d at 30 0 for
timre sufficient to allow 4-5 generations .
This provided
ade quate differentiation between live and dea d organisms .
Representative slide-cultures from populations with high
and low viabilitie s are illustrated in Fig . 2 .
S . lactis
ML 3 occurre d mainly as diplococci but chains with up to
six cocci we re observe d .
Slide-cultures we re counte d in
green light using a phase contrast microscope with an
eyepie ce grid (magnification x 3 1 0 ) .
As a routine , eight
typical fie lds with 40-60 obj ects/field were counted on
duplicate slide-culture s , significant overcrowding of
For slide-culture s of organisms
microcolonie s did not re sult .
with long division lags ( see Fig . 1 5a ) the incubation time was
increase d appropri.ately and ten fields were counte d with
20-3 5 obj e cts/field to minimize errors from overgrowth of
dead organisms .
Conventional colony counts were made by the
standard pour-plate method ( Cruickshank, 1 9 6 5 ) using 0 . 1/ 100
or 1/ 1 00 serial dilutions in phosphate buffer and the agar
medium de scribed above .
Plate s were incubate d until a
constant count was obtained, normally for 2 4-48 hours .
Cell division lag time s were measure d by a method similar
to that de scribe d by Postgate & Hunte r ( 1 9 6 3b , 1 9 6 4 ) .
Populat­
ions were starve d in various systems and at inte rvals sample s
Representative slide-cultures from starved populations
Fig . 2 .
Organi sms were starved at 30 0
of str epto coccus lactis ML 3 .
in O . 07 SM-pho sphate buff er , containing l0j" M-EDTA and lmM-MgS0 4 .
Slide-cultures for viabi lity deterniination were prepared
after 2 hr . ( a ) and 2 4 hr . ( b ) starvation and incubated at 30 0
for 3 hr . and 5 hr . r espectively .
The viability wa s determined
and typi cal fi elds photographed with a pha se contrast mi croscope .
Viabilities wer e 9 9% ( a ) and 20% ( b ) .
A
B
45.
were remove d, centrifuge d and the packe d organisms resuspende d
in broth me dium at 30 0 .
The viability of the population was
imme diately determined by slide -culture and its turbidity
measure d at intervals .
From the extrapolation of a semi­
logarithmic plot of turbidity incre ase to the turbidity of
the viable proportion of the original innoculum, an e stimate
of the division lag time was obtaine d ( see Fig. 1 5b ) .
ANALYTICAL METHODS
All colorimetric and spectrophotometric me asurements in
the visible and ultraviolet ( u . v . ) region were made with
either a Beckman DB or a Ze iss PMQII spe ctrophotometer using
l cm . glass or silica cells .
Magnesium and coppe r . The magnesium and coppe r contents of
cell-free supernatant fluids and buffer solutions were
measure d using a Te chtron AA3 atomic absorption spe ctro­
photometer ( Techtron Pty . Ltd . , Australia ) under standard
operating conditions .
Standard magnesium solutions and
samples containe d in phosphate buffer were dilute d 1/ 1 0
with deionized wate r t o appropriate concentrations .
Phosphorus interference was suppressed by using lanthanum
chloride .
Cellular magnesium content was determined by
the meth6.d of Webb ( 1 9 6 6 ) .
Perchloric acid was adde d to
copper standards and samples to a final concentration of
1 0% ( v/ v ) . Ammonium pyrrolidine dithiocarbamate solution
and methyl isobutyl-ketone were adde d ( Allan , 1 9 6 1 ) , and
the copper was determine d in the organic phase after shaking.
' Reserve ' polymers .
Polyglucose and poly-f -hydroxybutyrate
were assaye d by the methods of Strange et al e ( 1 9 6 1 ) and
Williamson & Wilkinson ( 1 9 5 8 ) re spectively .
Manometry .
Conventional manometric methods were use d
46 .
( Umbre lt , Burris & Stauffer, 1 9 5 7 ) .
Oxygen uptake was
determine d at 3 0 0 with a shaking rate of 50 oscillations/min .
P rotein . Prote in was determined by the biuret ' method
( Stickland, 1 9 5 1 ) .
Suspensions of S . lactis require d
heating at 1 0 0 0 for 20 min. in 0 . 7 5N-NaOH for maximum colour
development .
Supernatant buffer and standard sample s were
It was found that heating produce d higher
not heated .
blank readings so the appropriate corre ctions were use d .
Drie d bovine serum albumin ( Sigma , Agrade ) , containing 1 3 . 6%N ,
was used as the standard .
The alternative method of Lowry,
Rosebrough, Farr & Randall ( 1 9 5 1 ) gave similar results to
the biuret method with sample s of both supernatant and
alkali-hydrolysed cell suspension .
The reproducibility of the biuret method was teste d by
performing twelve analyses on a suspension of 1 . 0mg . dry wt .
bacteria/mI .
The mean protein content and standard
deviation was 0 . 4 8 ± 0 . 0 2 8mg . /ml .
Total nitrogen . Total ce ll N and supernatant N were determine d
by the micro-Kj eldahl method de scribed by Humphrie s ( 1 9 5 6 )
using a Se/K 2 S0 4 catalyst and methyl red-methylene blue
indicator .
Enzyme assays . Supernatant protein was assaye d for
tributyrinase activity ( see Lawrence , Fryer & Re iter, ; 1 9 6 7 )
and for proteolytic activity by the Folin-Ciocalteu method
( see Cowman & Speck 1 9 6 7 J .
Lactic dehydrogenase was assayed using the method
described by Kornberg ( 1 9 5 5 ) .
Supernatant samples ( 2 . 8ml . )
were adj uste d to pH 7 . 4 , 1 0mM-Na pyruvate ( O . lml . ) and
4mM-nicotinamide-adenine-dinucleotide ( NADH , O . lml . ) were
adde d and the rate of oxidation of NADH at 3 0 0 was determine d
using a recording spe ctrophotometer at 340 m� . The unit of
activity was taken as the amount of enzyme which cause d an
initial rate of oxidation of l ;u mole NADH per minute .
� .-Galactosidase
47 .
activity of supernatant sample s wa S'
teste d for by the o-nitrophenyl-j3 -D-galactopyranoside hydrolysis method as de scribed by Citti et al e ( 1 9 6 5 ) .
The assay for glycolytic activity de velope d in the
present inve stigation is de scribe d later in the Methods .
Amino acids and ammonia . Amino acids in bacterial extracts
and supernatant samples were measure d colorimetrically by
the ninhydrin method of Yemm & Cocking ( 1 9 5 5 ) .
Glycine
standards were use d and (NH 4 ) 2 S0 4 standards were included
to corre ct for NH 3 , which was determined separately by
the micro-diffusion method of Conway ( 19 4 7 ) .
Buffer
samples were adj usted to pH 5 . 0 before amino acid analysis
by addition of HCI or dilution in citrate buffer ( pH 5 . 0 ) .
Amino acids were separate d by thin-layer chromatography
( TLC ) on mixe d cellulose-silica gel layers and dete cted with
ninhydrin ( Turner & Redgwell , 1 9 6 6 ) .
Plate s were develope d
with phenol-wate r (.8 0 : 2 0 , w/v ) in the first direction ( 5 hr . )
and drie d overnight at 40 - 50 0 •
Butanol : acetic acid : water
( 5 : 1 : 4 , v/v/v, upper phase ) was use d in the second dire ction
( 4 hr . ) .
Two-dimensional , descending paper chromatography of the
amino acids in bacterial extract and supernatant sample s was
performe d at 2 2 0 using Whatman no . l paper and the procedures
of Roberts , Abelson , Cowie , Bolton & Britten ( 1 9 5 7 ) .
Supernatant sample s were de salte d by the ion-exchange
methods of Dre ze , Moore & Bigwood ( 1 9 54 ) .
Paper
chromatography solvents were ( i ) first direction ( 1 6 hr . ) ;
sec-butanol : formic acid : water ( 7 : 1 : 2 , v/v/v) ; ( ii )
se cond direction ( 1 8 hr . ) ; phenol : aq . NH 3 ( sp . gr . 0 . 8 8 )
water ( 8 0 : 0 . 3 : 2 0 , w/v/v) .
Papers were dried, dippe d in
acetone containing ninhydrin ( 0 . 2 5% ) and heate d at 1 0 0 0 for
Amino acids on thin-layer
5 min (Toennie s & Kolb , 19 5 1 ) �
and paper chromatograms were tentatively identifie d by
48 .
comparison with RF value s of standards .
More positive identification and quantitative measurement
of amino acids was obtaine d using a Beckman model 1 20C
automatic amino acid analyser with Be ckman custom sphe rical
ion exchange re sin .
The short column for basic amino acids
The elution
containe d resin type PA 3 5 to a height of 8 cm .
The long
buffer pH was 5 . 2 8 and. the flow rate 6 8ml . /hr .
column for acidic and neutral amino acids containe d resin
type PA 2 8 to a he ight of 5 8 cm .
Buffer, pH 3 . 2 8 , was use d
as eluant being replace d after 9 0 min . by a se cond buffer
at pH 4 . 2 5 . The ninhydrin flow rate was 34ml . /hr . and
analyses took place over a 4 hr . period .
Peaks were
identified from standard elution time s and integ�te d by the
height-width method .
Using standard calibration constants
the amino acids were estimate d with an accuracy of ± 1%
for the maj or peaks .
The total ce llular amino acid composition was determine d
on organisms harve ste d at the end of the growth phase and
Washe d organisms were
washed twice in distille d water .
free ze-dried, hydrolysed ( 6N-HC:l , 24 hr . , 1 1 0 0 in vacuum
sealed tube ) and the hydrolysate analyse d .
Extraction of soluble intracellular compounds . The complete
extraction of water soluble intracellular compounds from
bacteria is probably best achie ve d by heating the organisms
in boiling water ( see Holden, 1 9 6 2 ) .
The minimum he ating
time at 1 0 0 0 for complete release of the amino acid and
nucle otide pools of s . lactis was found to be 1 2 ± 3 min .
For routine extracts , bacteria were washe d and resuspended
in de ionize d water and the suspensions heate d for 20 min. at
1 0 0 0 in stoppered tubes .
Cell debris was remove d by
centrifugation and the clear extract pipette d off .
A second
extraction yielde d only a further 1 - 3% of ninhydrin-positive
material and was therefore not carrie d out routinely .
49 .
Ribonucle ic acid . Bacterial RNA was determine d by the method
describe d by Munro & Fle ck ( 1 9 6 6 ) .
Perchloric acid-washe d
bacteria ( 1 0 min . at 0 0 in 0 . 5N-HCI0 4 ) were subjecte d to
alkaline hydrolysis in 0 . 3N-KOH at 3 7 0 for 60 min .
This
was shown to give a complete extraction of ribonucleotide s .
( For convenience , sample s and standards were often store d
at _20 0 in alkaline suspension before hydro�ysis .
It was
shown that storage for up to 24 hr . at _20 0 did not affect
analyse s ) .
The hydrolysate was chilled to 0 0 and iceAfter
cold HCI0 4 was adde d to a final concentration of 0 . 5N .
1 0 min . at 0 0 the insoluble fraction was sedimented and
washed twice in ice-cold HCI0 4 ( 0 . 5N ) . Centrifugation was
carried out in a Sorvall refrigerate d centrifuge ( 30 , 9 0 0gj 1 min . )
The alkali extract and washings were combine d and made up to
2 5ml .
Sample s were filtered through a sintered glass filter
( porosity 5/ 3 ) before extinction measurement at 2 60 � .
Soluble yeast RNA ( Sigma , type III ) was use d a s a standard
and was treated in the same way as samples .
The standard
RNA containe d 7 . 8 5% phosphorus and was assume d to be 8 3%
RNA ( see Strange et al . , 1 9 6 1 ) . The method was highly
reproducible ; twe lve analyses on a suspension with 1 . 0mg .
dry wt . bacteria/mI . gave a mean RNA content with standard
deviation of 0 . 1 9 5 ± . 0 0 2mg . RNA/mI .
Deoxyribonucleic acid . Bacterial DNA was extracte d and
estimated by the methods of Burton ( 1 9 56 ) .
Deoxyribose
was use d as the standard .
Before measuring the
Extracellular u . v . Mabsorbing compounds .
extin�tion of supernatant samples at 2 5 7 � , the solutions were
deprote inized with 5% TCA ; the appropriate blanks were include d .
Tentative identification of the u . v . -absorbing compounds ,
which were released from starve d S . lactis organisms , was
e stablishe d by the following procedure s .
Supernatant
sample s were applied in bands to preparative TLC plates
50 .
(MN-Ce llulose , 300G) and chromatographed in deionize d water
following the method of Randerath ( 1 9 6 4 ) .
The bands , which
were distinctly separate d, were marke d under u � v . light ,
remove d and the u . v . -absorbing material elute d with three
portions of 0 . lN-HCl0 4 .
Afte r filtration through sintere d
glass ( porosity 5/3 ) , the u . v . spe ctra of the eluate s were
recorde d on a Be ckman DK-2A ratio recording spe ctrophoto­
meter along with spe ctra of standards in 0 . lN-HC 10 4 •
The RF value s and spe ctra of the unknown compounds were
compare d with those of standards and literature data
( Dawson , Elliott , Elliott & Jones , 1 9 59 ) .
Lactose . Lactose was estimated in cell-free sample s of
growth me dium by the anthrone method of Richards ( 1 9 59 ) .
Ribose . Free and purine-bound ribose was e stimate d in
cell-free supernatant or alkali-soluble nucleotide
extracts using the orcinol method (Mej baum, 1 9 3 9 ) .
Hexo se . Total ce llular hexose was estimate d as glucose by
the anthrone proce dure of Tre velyan & Harrison ( 1 9 5 2 ) or
by the re ducing sugar method of Nelson ( 1 9 44 ) .
For
comparison of the glycolytic activity assay ( describe d
late r ) with chemical analyses , glucose was determine d by
the more pre cise neocuproine method of Dygert , Li , Florida
& Thoma ( 1 9 6 5 ) ( se e Table 4 ) .
Lactic aci d . Lactic acid was measure d in deproteinize d
supernatant sample s b y the method o f Barke r & Summ e rson
( 194 1 ) .
Steam-volatile acids . These compounds were separate d by
exhaustive steam distillation in a Markham still, after
adj usting the supernatant and standard samples to pH 1
Distillates were titrate d with O . OIN-NaOH in
with H 2 S0 4 •
a stream of ' dry ' N 2 , using phenolphthalein as the
indicator .
51 .
Phosphorus . Phosphorus was e stimate d by the method of Burton
& Petersen ( 1 9 60 ) .
Extraction of a 50g. sample of casamino acids with
Lipids .
diethyl ether for 30 hr . in a soxhlet apparatus , yie lde d
Howe ver , all of this material
1 5mg . of semi-solid material .
was removed by washing with aqueous CaCl 2 ( Folch, Lees &
Stanley , 1 9 5 7 ) confirming that there was no appreciable
lipoid material introduced into the routine " growth medium
from the casein hydrolysate .
Similar observations were
reporte d by Ikawa ( 1 9 6 3 ) and Vorbeck & Marinetti ( 1 9 6 5 ) .
Lipids were extracte d directly fro� washed organisms by
the methanol-chloroform procedure of Vorbe ck & Marinetti
( 1965) .
A second extraction recove re d a further 6 - 9%
of the total lipid, and acid hydrolysis ( 2 . 5N-HCI , 16 hr .
at 1 0 0 0 ) of the re sidue yielde d a further 3-4% of the total
Two methanol-chloroform extractions were normally
lipid .
performe d .
Extracts were washed with aqueous CaCl 2
( Folch et al . , 1 9 5 7 ) to remove non-lipid material and the
/
solvents were remove d using a rotary e vaporator at 40 0 .
The tared flasks were finally dried to constant weight in
a vacuum desiccator over P 2 0 5 and the lipids were e stimate d
gravimetrically .
Silicic acid column chromatography was use d to separate
lipids into neutral and polar fracti.ons (Wren , 1 9 60 ;
Hirsch & Ahrens, 1 9 5 8 ) .
Lipid ( 2 0 0mg. ) was applie d to
the top of the column ( 2 50 x 1 7mm . ) in the minimum amount
of chloroform-methanol ( 4 : 1 ) and the neutral lipid was
completely elute d with ethanol-free chloroform containing
1 % methanol ( 50 0ml . ) .
Polar lipid was then elute d complete ly
Column
using chloroform-methanol ( 2 : 1 , v/v, 300ml . ) .
separations were monitored routinely by both T LC and
phosphorus analyse s .
Fractions obtaine d were drie d and
weighe d as describe d previously .
52 .
Thin-layer chromatography was carrie d out on glass plates
with a layer of silicic acid ( silica gel G, Merck ) following
the proce dures of Mangold ( 1 9 6 1 ) .
Chromatograms , which had
been deve lope d in either hexane : diethyl ether : acetic acid
( 70 : 3 0 : 1 , v/v/v) or diisobutyl ketone : acetic acid : water
( 8 0 : 50 : 8 , v/v/v ) , were drie d before spraying with ninhydrin
and the spots giving a positive reaction were marked .
Phosphorus-containing components were dete cte d by using the
molybdenum spray reagent of Dittmer & Le ster ( 1 9 6 4 ) .
Finally the plate was spraye d with 10% H 2 S0 4 and charre d
at 1 1 0 o to show all components .
The presence of a glycolipid was demonstrate d by refluxing a sample eluted from a
preparative TLC plate in 3ml . 2N-H i S0 4 for 1 hr . , extracting
the lipid re sidues with petroleum ether and neutralizing the
a que ous phase with barium carbonate .
After centriguation ,
the supernatant was removed and evaporate d to dryness at
reduce d temperature and pre ssure .
Re sidual sugars we re
redissolve d in water and samples applie d to Whatman no . 1
paper .
P apers we re/ develope d in ethyl acetate : pyridine :
water ( 2 : 1 : 2 : , v/ v/v) by the procedure of Jermyn &
Isherwood ( 1 9 4 9 ) and spraye d with alkaline silver nitrate
( Trevelyan , P rocte r & Harrison , 1 9 50 ) .
Glyceride s we re hydrolyse d by refluxing with aque ous
5N-KOH : CH 3 0H ( 1 : 1 , v/v) for 5 hr . ; non-saponifiable
material was removed by ether extraction .
After acid­
ification, free fatty acids were extracte d into _ �iethyl
ether, dried over anhydrous Na 2 S0 4 and e sterifie d with
diazomethane ( Schlenk & Gellerman ( 1 9 60 ) ) .
Analysis of the methyl e sters was carried out on an
Aerograph 600 gas chromatograph (Wilkins Instrument &
Re search, U . S . A . ) using a hydrogen flame ionization
dete ctor .
Apie zon L on 8 0 - 1 0 0 me sh celite ( 1 0%, w/w )
a t 1 9 6 0 and polyethy�ene glycol adipate a t 1 8 0 0 were
used as stationary phases and the columns were prepare d
a s describe d by Jame s ( 1 9 60 ) .
The dete ctor and columns
,
53 .
were che cked periodically with the fatty acid standards and
procedure s recommended by Horning, Ahrens , Lipsky, Mattson ,
Mead, Turner & Goldwater ( 1 9 6 4 ) .
Peaks were identifie d by
comparison with retention data of standards or with publishe d
values and the percentage composition of the fatty acids
was e stimate d by the heiglit-width method ( Horning et al . ,
1964 ) .
Solid reagents for analytical procedure s were
Materials .
recrystallized and solv-ents re distille d if analytical
reagent grade was not available .
All water was distille d
and then deionize d by passage through a mixe d bed
ion-exchange resin (Permutit ' Biodeminrolit ' ) .
Measurement of Protein Synthe sis . Pro�ein synthe sis in
starve d organisms was e stimated by fullowing the incorporation
of valine_ 1 4 C into cell protein using the procedure s outline d
by Willetts ( 1 9 6 7 ) .
Culture sample s ( 2ml . ) were adde d to
an e qual volume of trichloroacetic acid ( rCA , 10%, w/vJ and,
after heating. for 30 min . at 9 0 0 ( see Marchesi � Kennell ,
1 9 6 7 ) , the precipitates were filtered off on membrane
filters ( 2 5mm . , pore size 0 . 4 5� ; Millipore Filter Corp . ,
U. S.A. ) .
Each filter was washe d successively with three
1ml . volumes of TCA ( 5% , w/ v) containing DL-valine ( 1 50;-< g .!
Material
mI . ) , TCA ( 5% , w/v ) alone and acetic acid ( 1% ) .
isolated by this proce dure may include cell wall substance
as well as ' true ' protein .
Howeve r , lactic acid bacteria
contain little valine in the cell walls ( Ikawa & Snel l ,
1 9 6 0 ) and the proce dure seeme d t o b e ade quate for the
purpose of the pre sent investigation .
For the me asurement of valine _ 1 4 C uptake by whole cells,
culture samples ( 2ml . ) were filtered on membrane filters,
washe d with ten 2ml . volumes of 0 . 0 7 SM-phosphate buffer
The washing
containing L-valine ( 2 0 0� g . /ml . ) and drie d .
proce dure s use d for incorporation and uptake me asurements
54 .
remove d all radioactivity from control systems without cells
and the reproducibility of the measurements is shown in
Table 4 .
The radioactivity on the drie d membrane filter discs was
measure d with a Packard Tri-Ca�b 2000 series liquid
scintillation spe ctrometer ( gain settings 1 0%, window
settings 50- 1 000 ) with an efficiency of 70 . 5% as determine d
by channels ratio measurements .
The scintillation
solution consiste d of 2 , 5-diphenyloxa zole ( 5 . 0 g . ) and
1 , 4-bis-2- ( 4-methyl- 5-phenyloxa zolyl ) -benzene ( 0 . 3 g . ) per
litre of toluene .
GLYCOLYTIC ACTIVITY ASSAY
In view of the correlation between bacterial survival
and the activity of the enzymes catabolizing the energy
source ( see Introduction ) , it seemed appropriate to examine
the glycolytic activity of starved S . lactis organisms
since glycolysis repre sents the only maj or catabolic
pathway in this organism .
Existing methods for determining
glycolytic activity are based on the measurement of lactate
formation rates e ither by direct titration , manometric
technique s , colorimetric or enzymic analyses .
Howeve r ,
the se methods were either too insensitive , inaccurate or
laborious for the purposes of the present investigation .
The assay develope d involve s the measurement of the
radioactive anionic products ( lactate , acetate and
formate ) and the remaining unfermente d labelle d substrate
( glucose ) by liquid scintillation counting following
separation on DEAE-cellulose paper strips .
Materials . DEAB-cellulose paper ( DE8 1 , free base form ) was
obtained from Whatman , England .
D-Glucose-U- 1 4 C ,
sodium-DL-Iactate-2- 1 4 C, sodium acetate-2- 1 4 C, sodium
formate'- 1 4 C and glycerol_ 1 _ 1 4 C, were obtained from The
55 .
Table 4 .
Reproducibility of valine- 1 4 C incorporation and
uptake measurements .
Washe d organisms were resuspende d at
0 . 9 1mg . dry wt . /ml . in four 20ml . volume s of 0 . 0 7 5M-phosphate
buffer ( pH 7 . 0 ; containing 1 0� M-EDTA , 1mM-MgS0 4 ,
DL-valine- 1 - 1 4 C ( . 2 5 ;u c . /ml . , 6 u g . /ml . ) , L-valine
;
After incubation for
( 20 0 � g . /ml . ) and 1 0mM-D-glucose ) .
o
1 hr . at 3 0 , two 2ml . samples were remove d from e ach
culture and the valine _ 1 4 C uptake by cells determine d
( see text ) .
The remainder of the culture s was cooled
rapidly to _ 1 0 0 , four 2ml . sample s were remove d from
lnt 0 ce 1 1
, - 1 4 C lncorpora
'
t lon
"
eac h cuIt ure and the va I ine
protein e stimated .
Results are given as the mean +
standard de viation , with the number 'o f measurements in
parenthe se s .
Valine- 1 4 C in�orporation and uptake
( c . p . m . /mg . dry wt . bacteria/hr . )
P rotein
Cells
8 44
+
37
( 16)
3096
+
189
( 8)
56 .
Radiochemical Centre , Amersham, England .
Under the
influence of its own radiation , glucose-U- 1 4 C decompose s
in the free ze -drie d state at a rate of about 1 % per ye ar
at -20 o .
The decomposition products remain firmly bound
to DEAR-cellulose paper and cannot be elute d with
deionize d water .
To obtain a sample of glucose-U- 1 4 C
e ssentially free from de composition products, freeze-drie d
glucose-U- 1 4 C was dissolve d in a small volume of deionize d
water and applie d t o a DEAR-cellulose paper strip 2cm .
wide .
After running 1 0 cm . in deionized water by
a scending chromatography, the paper was dried, cut into
bands and elute d with de ionized wate r ; 9 8% of the
applied activity was elute d from the ' upper 4 x 2cm . of'
the paper .
This glucose-U- 1 4 C, free from anionic
impuritie s , was use d in subse quent experiments . Since
glucose fermentation by S . lactis may produce a variety
of end products and may proceed to a limited extent via
the HMP pathway ( see Introduction and Fig. l ) , it is
des irable to use a uniformly labe lle d substrate for this
assay .
The liquid scintillation solution consiste d of
2 , 5-diphenyl-oxa zole ( 5 . 0g . ) and 1 , 4-bis-2-( 4-methyl-5phenyloxa zolyl ) -benzene ( O . 3g . ) per litre of toluene .
Routine counting procedure . Standard radioactive compounds
were use d in preliminary experiments .
Drie d DRAE­
cellulose strips ( 2 x 2 . 5cm . , fol de d where ne cessary )
with a dsorbed labelled compounds we re placed vertically in
standard counting vials .
Scintillation solution ( 1 0ml . )
was added and vials were counted in a Packard Tri-Carb
2 0 0 0 serie s liquid scintillation spe ctrometer with gain
settings of 1 0% and window settings of 50- 1 000 in both
channels .
The absolute counting efficiency for glucose­
4
1
U_ C and lactate-2- 1 4 C on DEAE-cellulose paper with the
57 .
above settings was 5 5 . 3%, determine d from channels ratio
me asurements on aque ous solutions of the two compounds
For
in toluene-ethanol scintillation solvent .
. most
4
labe lled samples at least 1 0 counts were recorde d and
sample s near background activity were counted for 2 0 min .
For the scintillation counter use d, the orientation of
the DEAE-cellulose papers had little effect on count rate ;
for discs lying flat on the bottom of the vial the
activities were 3% lower than the values obtaine d with
vertical strips .
Vertical pl,a cement of strips was
the refore adopte d routinely .
Removal of papers after
counting did not significantly increase the background
count showing that all the C 1 4 _ labelle d compounds use d
remaine d adsorbed to the DRAR-cellulose paper in the
scintillation solvent .
There was a linear relationship
between the applie d activity and the count rate and the
count rate was independent of the distribution of the C 1 4 _
activity on the DEAE-ce llulose paper .
When several
2 x 2 . 5cm . blank DEAR-cellulose strips were place d on
either side of a paper containing C 1 4 _ labelle d compounds ,
the count rate was unchange d showing that the paper was
transparent to photons .
Chromatographic separation of compounds . Using a number
of different solvents , adsorbe d glucose-U- 1 4 C could not
be washe d off DEAE-cellulose papers using technique s
describe d by She rman ( 1 9 6 3 ) without removing much of the
a dsorbed lactate- 1 4 C .
It was therefore necessary to
1
4
separate glucose- C from the �nionic fermentation
products by chromatography and deionize d water was found
to be the most efficient solvent .
Sample s of solutions
1
4
containing C _ labelled compounds were applie d to
2 x 1 5 cm . DEAE-cellulose strips in a narrow band .
These sample s were not dried following the observation
58 .
that irradiation from infra-re d lamps re sulte d in a
sub stantial bre akdown of the glucose-U- 1 4 C to products
which remained firmly bound to the paper and sub­
se quently interfered with assays .
The paper strips
were elute d with deionized water by ascending
chromat-ography , the sol vent front being allowe d to
ascend 1 0 cm . from the sample ban d .
After drying unde r
an infra-red lamp the strip was cut along pre-rule d
lines , normally into 2 x Scm . strips , folde d when
ne cessary and placed vertically in counting vials .
Glucose-U- 1 4 C travelled at the solvent front while
lactate - 1 4 C remaine d at the origin ( Fig. 3 ) .
The pre sence of salts in the sample applied to the
DEAB-cellulose strip had a pronounce d effect on the
chromatographic separation of glucose - 1 4 C from
When 20-50 �1 .
lactate- 1 4 C in deionize d water .
samples in 0 . 0 7 5M-phosphate buffer were applie d to
.
2 x 1 5 cm . DEAB-ce llulose strips, the lactate- 1 4 C
tende d to move away from the origin and separation
from glucose- 1 4 C was not precise .
With 1 0 � 1 . sample s
in 0 . 0 7 5M-phosphate buffer the re was no salt effect .
Effe ct of metabolic products other than lactate . Sodium
acetate-2- 1 4 C and sodium formate- 1 4 C, dissolved in
phosphate buffer ( pH 7 . 0 ) , were spotted on separate
DEAE-ce llulose strips which were chromatographe d,
drie d, cut up and counte d as usual .
All the applie d
activity remaine d at the origin so that any acetate or
fQ'rmate produce d during glucose fermentation in
routine assays was counte d together with lactate .
Glyce rol- 1 - 1 4 C was found to travel at the solvent front
on DEAB-cellulose paper strips .
Routine assay procedure . For routine glycolytic activity
assays of starve d S . lactis organisms, fermentation systems
.solvent
front
lOcm
1602
1
1600
2
o
3
o
1
1
2
67 9
67 4
- - - -
Ocm
T
--
- - -
- - -
lL
lL
·
C lactate- C glucose- C
gluco se- lL
+l actate- l t
Fig . 3 . S eparation of standard D-glucos e-u- 1 4 C from standard
Na-DL-lactate- 2 - l 4 C on DEAE-cellulo s e paper . Samples ( 20 � 1 . )
of aqueous so lutions of glucose- 1 4 C ( O . 0 6 5 � c . /ml . ) and
lactate- 1 4 C ( O . 0 27 )l c . / ml . ) were spott ed on 2 x 1 5 cm . DEAEAfter elution
cellulose strips as shown by the cross ed area .
with deionized wat er by ascending chromatography , the strips were
dried , cut into four 2 x 2 . 5 cm . strips along pre-ruled lines , and
Counts are gi ven in c . p . m . minus background , figure to
count ed .
scal e .
59 .
contained ; D-glucose-U- 1 4 C ( 1 . 6 6x 1 0 3 c . p . m . / 1 0� 1 . ) ,
1 0mM ; MgS0 4 , 2mM; 0 . 0 7 5M-phosphate buffer , pH 7 . 0 ; and
organisms , approximate ly 0 . 5mg . dry wt . /ml .
Two samples
were remove d at time s designe d to give measurements in
the range of 20-60% glucose utili zation .
Each sample
was place d on a 2 x 1 5cm . DEAE-cellulose paper strip and
chromatographe d in de ionized wate r .
When the solvent
front had ascende d 1 0 cm . the paper was drie d, two 2 x Scm .
portions containing the labelled compounds were cut out
and counte d .
The mean glycolytic activity of the two
samples was expre ssed as �moles l actate/�g . dry wt .
bacteria/min .
The change in buffer pH after 20-60%
glucose utilization was less than 0 . 1 4 units .
4
Time course of glycolysis . The conversion of glucose-U= 1-. C
to lactate- 1 4 C by whole cells of S . lactis took place at a
linear rate until almost all of the .glucose was fermented
( Fig. 4 ) .
This rate produced 0 . 2 9 5� mole s lactate/mg . dry
wt . bacteria/min .
With a bacterial mass e quivalent to
0 . 69mg . dry wt . /ml . , 1 . 5-2 . 0% of the total activity
remaine d ce ll-bound during glycolysis .
Most of this
activity was released when all of the glucose had been
fermente d .
In routine assays of glycolytic activity
with bacterial masses similar t o that given above ,
bacteria were not remove d from samples prior to the assay
of glycolytic activity so that cell-bound activity was
measure d as lactate .
Relationship between bacterial mass and glycolytic activity .
glycolytic activity of S . lactis was directly proportional
to bacterial mass when me asure d in suspensions ranging from
0 . 1 1- 1 . 9 4mg. dry wt . bacteria/mI . ( Fig. 5 ) .
Low bacterial
masses may be assaye d more accurately by increasing the
specific activity of the glucose-U- 1 4 C .
Effect of glucose concentration .
With bacterial masses of
The
100
�
5
7
1
o
W
N
-1
I­
:::>
W
If)
o
U
:::>
-1
(9
TIME
Fig . 4 .
120
(min )
Tim e cour s e o f glyco lysis .
Organisms from the end
of the growth pha s e were wa sh ed and r esuspended in 10ml .
0
pho sphate buff er at 3 0 ( O . 0 7 5M-N a HP0 -KH P0 , pH 7 . 0 ,
2
2 4
4
An aliquot of the washed
containing 1 0/ M- EDTA + lmM-MgS0 ) .
4
14
suspen sion ( 5 . 7ml . ) wa s added t o 3 0 0�1 . of 0 . 2M-glucose- C +
0 . 0 2M-MgS0 s o lution to gi ve final concentration s ; 1 0mM-gluco s e­
4
14
C ( 1 6 0 0 c . p . m . / lO �l . ) , 2mM-MgS0 , and 0 . 6 9 mg . dry wt .
4
ba cteri a/mI .
The suspension wa s incubated at 3 0
0
without a gitation .
At intervals , samples ( 0 . 5ml . ) were remo ved and immedi ately
0
chi l l ed to 0 •
A l i quots ( 1 0 J..ll . ) of th e suspen sion were
r emo ved with a syri nge and pl a c ed on DEA E - c e l lulose strips , th e
r em'ai ning chi l l ed suspension was c ent rifuged and 10 )'1 . samples
of the c e l l -free sup ernatant were pl a c ed on DEA E - c ellulose
strips .
Strips were chromatographed , dri e d , cut up and count ed
a s descri b ed in th e t ext ;
supernata,nt .
()
, cell suspension ;
[]
, c ell-fr ee
0-6�
0-5
o
1-0
BACTERIAL
MASS
1-5
2-0
( mg�y: wtiml )
Fig. 5 .
mass .
Relati onshi p b etween gl yco lytic qctivity and bacter i a l
Wa sh ed sus pensions of str epto co ccus lactis were prepa red
i n a simi l a r manner to tho s e desc ribed in
Fig. 4 .
A range of
bacterial conc entrati ons were pr epa red by di luti on in 0 . 0 7 5M­
pho sph ate buffer .
Di luted sampl es ( 1 . 9ml . ) wer e added to
14
1 0 0/ 1 . o f 0 . 2M-gluc o s e- C + 0 . 0 2M-MgS0 and th e tub es were
4
i ncubated at 3 0 0 •
Two sampl es ( 0 . 5ml . ) were r emoved at times
14
design ed to gi ve measurement s b etween 2 0 - 00 % gluc ose- C
uti l i zation .
A fter rapid chi l l i n g and cent ri fugati on at 0 0 ,
l 0 ;U l . sampl es o f the c e l l -free sup ernatants were placed on
DEA E- c ellulose st r i ps whi ch were a s sayed as desc ribed i n the
. t ext .
60 .
0 . 6 6 and 0 . 1 3mg . dry wt . /ml . , a constant glycolytic
activity ( 0 . 2 9� moles lactate/mg . dry wt . bacteria/min . )
was observe d with glucose concentrations in the range
0 . 6 7mM to 1 0 0mM .
Each system containe d the same amount
This re sult would be consistent with
of radioactivity .
the pre sence of an active transport system for glucose
which was not rate-limiting.
Comparison of isotopic assay with ghemical analyse s . The
glycolytic activity determine d separately by chemical
analyses on the rates of glucose utilization and lactate
formation was in agreement with results of the isotopic
assay ( Table 5 ) .
The maj or advantage s of this assay over existing
methods are that as well as being rapid, sensitive and
highly reproducible , it measure s simultaneously the rates
of glucose utilization and lactate formation in a single
operation .
All anionic products ( lactate , acetate ,
formate etc . , ) are measure d together , while any non­
anionic products ( e . g . glycerol ) are measure d as glucose ,
resulting in l ower assay .
Calculation of any loss of
radioactivity from the system ( total activity minus glucose
activity minus lactate ) would provide a measurement of
volatile product formation ( ethanol , CO 2 etc . ) .
Hence
in a single assay of glycolytic activity, considerable
information can also be provided about the glucose
fermentation products of a microorganism .
Incorporation
of label into ce ll components may result in loss of coUnts
Howe ver , only a small percentage of
from some systems .
the total activity was cell-bound at normal cell densitie s
of starve d S . lactis and most of this was released when all
the glucose had been fermente d .
This method would appear to be much more rapid, efficient
and pre cise than the Sephadex column method re cently described
by Riley ( 1 9 6 8 ) for the separation and . measurement of C 1 4 _
labelle d glucose and lactate .
61.
Table S .
Comparison of isotopic assay with chemical
Washe d suspension ( S Oml . ) was prepare d from S Oml .
analyses .
growth culture as for Fig . 4 .
Final concentrations were
1
4
1 0mM-glucose- C , 2mM-MgS0 4 , bacterial mas s 0 . S4mg . dry
wt . /ml .
Sample s ( Sml . ) were remove d at intervals ,
rapidly chille d to d O , centrifuge d and the cell-free
supernatants analyse d as below .
Values are given as the
mean ± standard deviation , with the number of independent
determinations given in parentheses .
Time
Isotopic assay
Glucose assay
Lactate assay
(min . )
% conve rsion
glucose �lactate
% glucose
utilization
% conve rsion
glucose� lactate
21
49
7S
100
1 60
1 8 . 0 6±. 1 7
40 . 7 2 ±. 2 6
6 1 . 04±. 2 8
8 1 . 6 0±. 2 4
9 7 . 4 6±. l S
(S)
(S)
(S)
(S)
(S)
2 0 . 6±. S
42 . 2± . 7
6 3 . S±. 4
8 3 . O±. 3
9 7 . 9±. 4
(6)
(6)
(6)
(6)
(6)
1 7 . 8±. 6
3 9 . S± . 8
6 1 . 7± . 9
7 8 . 9 ±. 6
9 3 . S±. 6
(6)
(6)
(6)
(6)
(6)
62 .
ELE CTRON MI CROSCOP Y
T o 2 0 mI . culture was added 2 mI . o f fixati ve ( 0 . 0 9 5M­
and 1 %
2
After mixing , the culture wa s c entri fuged
pho sphat e buffer , pH 6 . 1 , containing 6mM-MgC1
glutaraldehyd e ) .
( 1000g/ 5 min . ) and the o rgani sms r esusp ended in fixative ( 2 mI . )
o
and left for 3 hr . at 4 .
Fol lowing further c entri fugati on ,
the o rgani sms were washed twi c e in O . lM-pho sphat e buffer ( pH 6 . 1 )
containing 0 . 2M-sucro s e .
The pellet was then resusp ended in
O . lM-pho sphat e buffer containing 1% o smium t etroxi d e and l eft
0
After two further wash es in pho sphat e
for 2 hr . at 4 •
0
buffer th e o rgani sms were s et in 2 % agar at 4 5 •
The solidi fi ed
block wa s cut into small pieces and imm ersed in uranyl a c etate
( 0 . 5% , aq . ) for 2 hr . at room t emper atur e .
The agar blocks
were then d ehydrat ed in graded aqueous ethanol so lutions
acco rding to the fo llowing schedule :
9 5 % - 3 0 min . , 100%- 3 0 min . ( twi c e ) .
2 5 % - 1 5 min . , 7 5 %-1 6 hr . ( 4 ° ) ,
The blo cks wer e then
wa shed with propylene oxide and emb edded in araldite resin
using the metho d of Luft ( 1 9 6 1 ) .
Sections were cut with either a gla s s o r di amond knif e
using a L . K . B . ultrami crotome and pick ed up on copper grids
having either a co llodion-carbon suppo rting film o r no
suppo rting film .
S ections were stained by floating the gri ds
on lead salt so lutions ( Mil loni g , 1 9 6 1 ;
Reynolds , 1 9 6 3 ) for
1 min . fo llowed by wa shing with distilled water .
Some
s ections were float ed on 7 . 5 % H 0 for 5 min . prior to lead
2 2
staining to reduc e the d ensity o f the ba ckground cytoplasm and
thus inc r ea s e the contra st of ribosomes ( Silva , 1 9 6 7 ) .
Some
organisms wer e fi xed with o smium tetro xi de and postfixed with
uranyl a c etate a c cording to the method o f Kellenb erger , Ryt er
& S echand ( 1 9 5 8 ) .
These o rgani sms were then dehydrated and
emb edded as d escribed above and sections were treated with
Reyno ld ' s ( 1 9 6 3 ) l ea d stain .
S ections wer e examined with a Phi lips EM2 00 electron
mi croscope equipped with a single condens er syst em and a
4�
obj ecti ve op erating at 60Kv .
EXPERIMENTAL
PART I .
SURVIVA L OF STREPTOCOCCUS LACTI S
Growth of the organism .
The physi ca l and chemi c a l properties
of the growth envi ronment not only determin e the growth rate
and chemi cal composition of bacteria but also profoundly affect
their sub sequent metabolism and survi val when sta rved
( Strange et a l . , 1 9 61 ) .
In the present study it was therefo re
nec essary to define the growth conditi ons for Streptoco ccus
l a ctis as completely as possible .
Use of a chemi ca l ly defined
medium wa s impra cticable for reasons a lready di scussed .
Preliminary experiments indi cated that an amino a ci d-limiting
medium produc ed lower growth rates and in vi ew of the reports
of unbalanced cell wa l l synthesi s and sub s equent lysis o f
Streptoco ccus fa ecalis in c ertain amino acid- limiting media
( Sho ckman , Conover , Ko lb , Phi l lips , Ri l ey & T o enni es , 1 9 61 ) ,
it was decided to use a l a ctose-limi ting medium .
The choi c e
of l a cto re app ears t o have been particul arly fortunate i n vi ew
of th e rec ent finding by Mousta f a & Collins
( 1 9 6 8 ) that growth
of lacti c streptoco cci on gluco s e resulted in lysi s .
It wa s
estab lished that gluco samine , whi ch was es s enti a l for complete
cell wa l l synthe si s , wa s formed from gala cto se but not from
gluco se .
With an o rganism such a s S . l a ctis , whi ch produced large
quantiti es of l a ctate during growth , some contro l o ver changing
medium pH wa s n ec essa ry .
Exp eriments were undertaken to
determine the l a cto se and buffer conc entrations requi red to
obtain an ad equate c el l yi eld at the maximum growth rate with
a minimum change in pH .
are li sted in T able
6.
Some results from typi c a l exp eriments
F rom the limited number o f exp eriments
undertaken it woul d appear that bacteri a l mass yi eld was
proporti onal to the amount of energy substrate used , as
postulated by Bauchop & E l sden
( 1 9 60 ) for ana erobic glyco lysis
( see Gunsalus & Shust er , 1 9 61 ) .
The t erm ' mo lar growth yi eld
co effi ci ent ( Y ) ' , has been defined as the grams dry wei ght of
65 .
The routin e
Growth o f Streptococcus l a cti s ML .
3
medium wa s us ed exc ept for variation o f the l a ctose and buffer
Table 6 .
components .
Exp erimenta l media ( 2 0 0ml . ) were inooulated with
growth pha s e organisms from a similar medium and incubated at
0
3 0 in a water bath .
Buffer
Lacto s e
(%)
1.0
0 . lM-p0
1.0
0 . 2M-P0
1.0
0 . lM-P0
0.5
pH
Initi a l Final
4
4
4
+0 . 0 8M-N aHc0
d
0 . 0 5M-P0
4
+0 . 0 5M-NaHc0
a -- p0
b
a
4
3
c
C e l l yi eld
( mg · lml . )
b
Gen eration
time ( min . )
1 . 09
52
7.0
4.6
7.0
5.3
1 . 21
79
7.3
6.4
1 · 32
61
7.3
6.3
0 . 65
46
3
buffer was Na HP0 -KH P 0 .
2 4
2
4
Mean time for doubling o f turbidity in logarithmi c
growth phas e .
c -- Growth limit ed by pH , other cultur es were l a cto s e- limited .
d
Routine medium .
66 .
organi sms pro duced per mo le o f substrate metabo li z ed
The mo lar growth yi eld coeffici ent C y
}
� g 1 uco s e ,
� for ana erobi c glycolysis
and the AT P yield co effi cient � Y
ATP
by S . faecalis wer e 2 1 . 0 ± 4 . 0 and 1 0 . 5 ± 2 . 0 respectively
( Monod , 1 9 4 9 ) .
( Bauchop & El sden , 1 9 6 0 ) .
Growth of S . lactis in the routin e
=
medium ga ve Y
47 whi ch is simi lar to th e value
l ac t o s e
reported f o r st r eptoco ccus di a c eti lactis ( H arvey & Collin s , 1 9 6 2 ) .
These autho rs considered that thi s value was consist ent with
the uti li zation of mo re than 9 5 % of the f erment ed lactose a s
a n energy sour c e , indicating that only a small amount o f la cto se
value for
This Y
l a c to s e
S . la ctis a lso suggests that the Embden -Meyerho f p athway i s
carbon is us ed for c ell synthesi s .
the only maj or pathway f o r ATP producti on in thi s organism
( s ee Oxenburgh & Sno swell , 1 9 6 5 ) .
Growth charact eri sti cs with the routin e medium final ly
adopted a r e shown in Fig . 6 .
in thi s medium .
Growth wa s highly r eproducible
The mean gen eration time ( doubling of
turbi dity ) of S . lactis ML
during the logarithmi c growth phas e
3
i n batch cultur e was 4 6 + 4 min .
Analyses showed that lacto s e
wa s t h e growth-limiting sub strate .
to 6 . 3 ± 0 . 1 at th e end o f growth and
T h e pH changed from 7 . 3
the c ell yi eld wa s
0 . 6 5 ± 0 . 0 3 mg . dry wt . organi sm/mI . corr esponding to about
9
5xl0 cocc i/mI .
B acterial numb ers in resuspended syst ems .
It wa s impo rtant
in the present investigation to det ermin e wh ether the total
c ell numb ers in a gi ven suspension r emained constant on
prolonged incubation .
Accordingly , total count s/mI . were
ma de as well a s vi ability count s by the method of Po stgate et al .
( 1 9 6 1 ) , whi ch d etermines only the ratio of vi able/total organisms .
Four washed susp ensions ( approx . 5 0;U g . dry wt . organi sms/mI . )
in pho sphate buf f er were sampled o ver a 2 8 hr . p eriod .
For
total chain counts , qua druplicate samples from one susp en si on
and singl e samples from the other susp ensi ons were counted .
The vi able chain counts gi ven in Table 7 a r e the means of plate
counts from samples of all flasks di lut ed in duplicate and
.il2.S1
_-0-----0
m
::l{.,
....
.... 1
I
(9
w
�
2.2
6.9
pH 6 ·
r
a::
0
..J
<l:
0::
w
....
1.6
(9
9
0
6.
medium .
2
4
HRS FROM I NNOCUL ATION
Growth o f Streptoco ccus l a ctis ML
o
....
6
3 in the routine
The medium wa s inoculated wi th log-phase cultur e :
() , bacteri a l mass ;
shown .
w
tf)
6-1
U
<l:
Q)
Fig .
].3 0.5
0-4
5 0·3
0.2 ..J�
0·1
[]
, l a cto s e concentrat ion ;
6
, pH are
F o r sub sequent experiment s , organi sms wer e ha rvested at
the point on the growth curve indi cated by the a rrow , un l ess
otherwi se speci fi ed .
67 .
plate d in duplicate
The number of
( i . e . , 1 6 plate s ) .
cocci / chain and the % viabil ity figure s from sl ide-culture s
are me ans of duplicate counts from e a ch suspension ( see Methods ) .
The re sults in Table
7 show that S . lactis ML 3 organisms
had a tendency to clump in phosphate buffer on prolonge d
storage .
This was the re ason f or the de cre ase in total
chain counts but the re was no significant de crease in t otal
numbers of cocci for at least
28 hr .
The incre ase in cell
numbers in starve d suspensions of Salmone lla typhimurium
( Schae chter, 1 9 6 1 ) and Ae robacter aerogene s ( De an , 1 9 6 7 ) was
not ob serve d with starve d S . lactis . The 2 0 % de cre ase in
turb idity was prob ably a re sult of endogenous metabolism and
leakage of ce llular material into the suspending buffe r .
The re appe are d t o be a c onstant systematic e rror in the
%
viability re sults obtaine d by normal methods , s imilar t o that
note d by Norris & P owe ll
( 1 9 6 1 ) , Postgate et al . ( 1 9 6 1 ) and
Strange et al .
The se l ow figures we re probably the
( 196 1 ) .
re sult of errors in the total count dete rmination , since the
survival curve s were ve ry similar when plate count viabilit ie s
were expre sse d as pe rcentages of the initial pl ate count
( taken as 9 9 % ) .
The significance of viability me asurements cannot be
accurate ly asse sse d unless they are a ccompanied by statistical
data .
The sl ide-culture techni que o f P ostgate et al .
( 19 6 1 )
Six
in the following way .
3
wa she d suspensions were prepare d in phosphate buffe r ( see
was e valuated for S . lactis ML
Methods ) .
Duplicate samples were remove d from e ach suspension
at t ime s de signed t o give measurements at high , inte rme diate
and l ow viabilitie s .
Slide �eparat ion and count ing we re
performe d as de scribed in Methods .
was carrie d out on the
time .
An
analysis of variance
% viabil ity f igure s for e ach sampling
The standard de viation from the mean wa s compute d
together with the
9 5% confidence inte rval for two sl ide s
prepare d from one suspension ( F i g . 7 ) .
At high viabilitie s
( 9 4-99%) ', variance between tubes was negligible and the
accuracy of the method was high but variance incre ased with
Table 7 .
constancy o f total c e l l numb ers and c omp a r i son o f survi va l mea surem ents on
str ept o c o c cus l a ct i s ML i n washed susp ensions at 3 0 o .
O r gani sms were h a r v e s t ed at the
3
end o f the growth pha s e , washed twi c e and resusp en d ed i n 0 . 0 7 5M - pho sph a t e buf f er ( pH 7 . 0 ,
conta ining 10
M-EDTA ) at approx . 5 0 ;u g . dry wt . o r gani sm/mI . with gent l e a gi tation
�
p r o vi ded by a magneti c stirrer .
Sus p ension i ncubati o n
time ( hr . )
1
3
5
7
10
24
28
1 . 10
( l ) T ot a l chain c ount
8
( xlO - Iml .)
1 . 52
l . 47
1 . 36
1 . 45
1 . 42
1 . 12
( 2 ) Vi ab l e chain count
8
( xl O - Iml .)
1 . 19
1 . 16
1 . 10
0 . 52
0 . 22
0 . 03
78
81
36
15
3
99 . 5
96 . 5
65
28
6
( 3 ) % Viabi lity (( 2 ¥( 1 ) x 1 0 0)
( 4 ) % Vi abi lity ( s l i d e ­
cultur e )
( 5 ) C o c ci/chain o r clump
( 6 ) T otal c o c c a l count
8
( ( 1 ) . ( 5 ) xlO - /m1 . )
3 . 88
0 . 17 3
( 7 ) Turbi dity
0 . 165
0 . 1 57
b
2 . 71
3 . 56
3 · 45
+ 0 . 0 5 ( 8)
3 ·93
3 . 99
3 . 79
+0 . 2 2
0 . 140
0 . 139
0 . 149
0 . 143
See Fig . 7 .
c -- Calcul a t e d from chain l ength distribution , co c c i / chain ( % total chai n s ) ;
2 ( 6 0% ) ,
3 ( 17% ) , 4 ( 1 5% ) ,
5 ( 5% ) ,
6 ( 1% ) .
a
+0 . 0 8 (1 6)
a -- Standa rd d evi ation ( no . o f mea sur ement s/sampl e ) .
b
S.D.
1 ( 2% ) ,
1 00 �r-
��_
__
__
__
__
75
>­
I..J
� 50
>
25
o
Fig . 7 .
3
TIME
( hrs )
6
9
Range of pos sib l e vi abi liti es for starved St reptococcus
using the slide-cultur e method .
Mean vi abi liti es , () ,
3
and th e va riance for 9 5% confidence limits with th e routine
l a ctis ' ML
counting pro c edur e are indicated by vertical bars .
se� t ext and , Methods .
For details
For a random di stribution of vi able cocci
among chains ,. th e estima ted individua l coccal vi abiliti es are
shown , 0 ( see t ext ) .
dying populations which had variable cell division t ime s .
Variance was large ly a function of division l a g time .
The viabil ity me asurements of Burleigh & Dawe s
( 1 9 6 7 ) on
Sarcina lute a , and probably those of many othe r worke rs ( f o r
example Tempe st et al . , 1 9 6 7 ) , we re complicate d by clumping
or aggregation of individual cells .
Assuming t he individual
cells in a clump a re independent and have varying c apacit ie s
for survival then an errone ously high e stimate of viability
is obtaine d with any dire ct growth method .
In this situati on
correlations between endogenous metabolism and sur vival are
ma de more difficult .
Similar problems occur with chain-forming
b a cteria and it is obviously preferable to determine the
numbers of individual viable cells rather than viable clumps
or chains .
Using the distribution o f chain lengths a fter 3 hr .
starvation ( T able
7 ) , estimate s o f the individual coccal
viability we re obtaine d using the probability e quations
de s cribed by Robertson
( 1968 ) .
A random distribution o f
viable cocci among chains w a s assume d .
The se e st imate s thus
repre sent the minimum possible viability value s of starve d
culture s ( Fig .
7) and the slide-culture viability represents
a maximum value ( see Burleigh & Dawe s , 1 9 6 7 ) .
The diffe rence
between the se extreme estimate s can be conside r able , e ven
with short chain organisms such as S . lactis ML 3 ( Fig .
7 ) but
the de ath curve was very steep in most subse quent expe riments .
Starve d S . l actis organisms showe d a slight tendency to clump
( T able 7 ) but the re sulting change in chain ( clump ) size
distribution ha d a ne gligible e ffect on the calculated
individual coccal viabilities for at le ast 7 hr .
If the
individual cocci in a chain have varying capacitie s for
survival , then a s a popul ation be gins t o die the de a d organisms
should have a highe r proportion of short chain s than the total
population .
Howe ve r , from obse rvations of
d'ying populat�ons
on slide -culture s it appe a re d that the distribution of chain
lengths 'of de a d organisms was ve ry similar to that of the
total population .
Attempts to obta in a statistical correlation
70 .
to substanti ate thi s observation were unsucces sful due to
the difficulty in differentiating between the individual cocci
of dead chains on slide- cultures .
Survi val in resuspended syst ems
Survi va l mea sur ement s on all resuspended syst ems were
r ep eat ed at lea st thr ee times .
Survi val curves showed small
fluctuations with diff er ent batches of o rganisms but t r ends
b etween syst ems were a lways the same .
Eff ect of ethy1enedi aminet etra-a c etate ( EDTA ) .
E a rly
experiments on survi val in phosphat e buffer ga ve vari ab l e
result s and rapid d eath rates whi ch were decrea s ed on additi o n
of EDTA ( Fig .
8) .
The minimum eff ecti ve EDTA concentration
wa s approximat e1y � .
High concentrati ons of EDTA ( 10 mM )
a c c el erated death , po ssibly a s a result o f its c apacity to
destabili z e cell walls ( Gray & Wi lkinson , 1 9 6 5 ;
Eagon , 1 9 6 6 ) or ribo somes (Wade , 1 9 61 ) .
Asbell &
I t seemed likely
that a toxic metal impurity was pres ent in the buffer
( Po stgate & Hunt er , 1 9 6 2 ) .
Extra ction of pho sphat e buffers
with o rgani c metal compl exing r eagent s ( s e e Methods ) and ana lysi s
by atomi c absorption sp ectro scopy showed a copper concentration
2+
When trace amounts of eu ( 1 0 Ji M )
of about 0 . 0 6 p.p. m . ( lJL M ) .
were add ed t o suspensions containing 5;U M- EDTA , the survival
curve wa s similar to that for buffer without EDTA ( F ig . 9 a ) .
2+
MacLeo d , Kuo & Gelinas ( 1 9 6 7 ) reported that contamin ant e u
in the plating diluent c aused met aboli c damage to A . a erogenes
r esulting in increased nutritional r equi r ements ( s e e also
Burke
&
McVeigh , 1 9 6 7 ) .
Effect of di val ent metal ions .
The effect of divalent metal
ions was studi ed with susp ensi ons containing sufficient EDTA
to r emo ve the toxi c eff ect of contaminant copper .
Mercuri c
ions ( l;U M ) caused complet e d eath within 5 min . whi le the
toxicity of other ions test ed decrea s ed in the o rd er
2+ >
2+ >
2 + > e 2+ > . Zn 2+ > Pb 2 + ( Fi g . 9 a ) .
eu
0
Fe
Ni
This appears to be the approximat e order o f d ecrea sing stability
of the metal-EDTA compl exes ( Perrin , 1 � 6 4 ) so that it is unlikely
75
,.....
�
0
'-'"
>I....J
-
aJ
<l
>
50
25
o
. Fig . S .
ML 3 .
2
TIME
4
( hrs)
6
Effect of EDTA on survi va l of Str eptococcus lactis
O rganisms were harvested at the end of the growth pha s e ,
wash ed and r esuspended i n 0 . 0 7 5M - pho sphate buffer a t appro x .
0
2 0� g . dry wt . /ml .
O rgani sms wer e incubated at 3 0 , pH
7 . 0 0 ± 0 . 0 5 , �ithout agitation ( for detai ls see Methods ) .
Buf fer contained th e following concentrations of EDTA : 6
2
· 0 , 5xlO - 6
0 , 10 - 6 M ;
1 0- M ;
, 10 - 5 M ;
, no EDTA .
M;
Vi abilities were determined by the slide-cultu� e method .
.
71 .
that any of these ions di splac ed the contaminant copper f rom
2+
2+
its complex .
Calcium ions, Mn , and Sn
(l
M and 10
M)
were without effect .
2+
Addi tion of Mg
produc ed greatly increased survi val times
2+
( Fig . 9b ) , maximum survival b eing afforded with about 100�M -Mg .
Magn esium ions also prolonged survival o f o rgani sms in pho sphate
buffer without EDTA .
When o rgani sms were incubated with
2+
2+
2+
Mg + Cu , the toxi c effect of Cu
wa s decrea s ed ( Fig . 9b ) .
�
�
These obs ervations are similar to tho s e made by MacLeod &
Snell ( 1 9 50 ) for L actobaci llus arabino sus and by Abelson &
Aldous ( 1 9 5 0 ) for Escheri chi a coli .
The experiments described
2+
show that added Mg
may produc e two effects : ( i ) a decrea s e
2+
i n Cu
toxi city and ( ii ) a s eparate eff ect dec r ea sing the
death rate .
Effect of bacterial concentration .
Very d ense suspensions
( equi v . 7 . 8 mg . dry wt . organism/mI . or about 6xlO l O cocci/mI . )
survived b est , with decreasing survi val times at lower
bacterial concentrations ( Fi g . lOa ) .
Measurements o f
2+
Mg
excretion by the organi sms ( see Methods ) , showed that
2+
l eakage from the
at high bact eri a l concentrations Mg
o rgani sms was sufficient to produc e protective concentrations
in the suspending buffer ( Fig . lOa ) .
Analyses of whole
bacteria immediately after washing in buffer indi cated 0 . 41%
2
(w/w ) magnesium .
The concentration o f excreted Mg + ( 7 0 0� M )
i n the suspending buffer containing a n equival ent bacterial
concentration of 7 . 8 mg . dry wt . /ml . , represented a loss of
0 . 2 2% (w/w ) magnesium f rom the organi sms ( i . e . 5 4% o f the
cellular magnesium ) .
F rom these results it seemed likely
2+
that Mg
excr etion by the organi sms was the caus e of extended
survival at high bacterial concentrations .
Additiona l suppo rt
for this conclusion was obtained in a similar experiment
2
where the addition of 1 0 0;U M-Mg + gave a lmost identical
survival curves at ea ch bacterial concentration (Fig . lOb ) .
High bacterial concentrations a lso prolonged survival in
phosphate buffer without EDTA , presumably b ecaus e the copper
"
H
toxi city wa s decrea s ed by the excreted Mg
in a similar
I
75
75
.,!!
o
>�
:J 50
dj "
>-
!::: 50
dj
-.J
�
>
<i
5>
25
o
25
4
2
TIME
(hrs)
6
o
10
TIME (hrs)
20
30
Eff ect o f diva l ent metal ions on survi val of
Cell suspensions were prepar ed
Streptoco ccus lactis ML 3 .
Tub es contained SxlO - S M-EDTA in O . 07 SM­
as fo r Fi g . 8 .
Part ( a ) : () , no additi on ;
pho sphate buffer .
+Pb ( N 0 3 ) 2 ; 0 , + ZnS0 ; (II , +CoC1 2 ; 6 , +F eS0 4 ; A
4
+NiC1 2 ; X , +CuS0 4 ;
all salts 1o - S M .
Part ( b ) : � ,
no addition ; r emain ing tubes contained MgS0 at
4
"
conc entrati ons ; • , 1o- 3 M ; () , lO - 4M; 0 , lO- SM ; iJ
4
lO - M + CUS0 ( I O - S M ) .
4
Fig . 9 .
100
100
8
}:
I
"
I
0
75
;1
6
(a)
X
;tl
75
Ol
}:I
=..
I
50
I
I
I
I
I
I
I
r
Z
I
4�
I
Z
a::
W
CL
:::>
til
9
2
25
..,.!!
�
>-
t::: 50
...J
(Xi
4:
:>
25
o
10
TIME (hr)
30
20
Fig . 10 .
Effect of c ell concentration on sur vival of
C ells were harvested from
streptococcus lacti s ML .
3
S O Oml . of culture at the end of the growth pha s e , wa shed and
r esuspended in 60ml . O . 07 SM-phosphate buffer with lO�M-EDTA .
,
Part ( a ) . Vi abilities of washed suspensions ( S Oml . ) containing
7 . 8mg . dry wt . ba cteri a/mI . , O . 7 7�g . /ml . , 0 . 0 7 8mg . /ml . , and
O . 0 1 9mg, . /ml '. , are shown ® , ./ , A , and
X r espectively .
2+
Supernatant Mg
concentrations at the same c ell densiti es
were 0 " 0 , D. , and X
At intervals viabi lity determinations
were made , after sample di lution wher e n ecessary, and S mI .
sampl es' withdrawn from each suspension .
The samples wer e
c entrifuged and the c ell - free supernatants car efully r emoved
and filtered through sintered glass ( porosity S ) into tub es .
These samples were fro z en and later analysed for magnesium
( s ee Methods ) .
Part ( b ) .
Cell suspensions were prepared
a s in Part ( a-) containing ;
D. , O . 0 7 9mg . /ml .
0 , 8 . 1 mg . /ml . , 0 , 0 . 8 0mg . /ml . ,
E ach suspension contained lOO�M -Mg
2+
.
72.
manner to that described in the preceding s ection .
The survival
Effect of growth phas e and media compo sition .
o f o rganisms taken f rom different growth phases in the lactos e­
limiting medium were compared .
O rganisms f rom the mid­
loga rithmic phase , the end of the growth phase and f rom 1 and
2 hr . after growth had ceased, showed no measura,ble differenc es
in the survi val curves .
This i s in agreement with the
obs ervations o f Postgate & Hunter , ( 1 9 6 2 ) who stressed the
importance o f: the nutritional status of the population and
suggested that for a genetically uniform popul ation the
growth phas e has only a small effect on the survival time
during starvation .
Attempts to find a sati sfactory nonWhen the
carbohydrate-limiting medium were unsucc es sful .
lacto s e concentration was increased to 1% , growth c eased
b ecause of the inhibitory acid conditions produced ( about
pH 4 . 7 ) ; analyses indicated the presence of 0 . 0 8% lactose
at the end of growth .
These organi sms , when washed and
resuspended , showed a slightly increa s ed death rat e compared
When the amino acid
with organisms from the routine medium .
concentration wa s decrea s ed from 0 . 5% to 0 . 0 5% the growth rate
was substantially decrea s ed ( doubling time 2 - 2 . 5 hr . compared
with about 4 6 min . for the routine medium ) , and resuspended
organi sms from thi s medium also showed higher death rates than
normal ( 5 0% decrease in viability in 4 . 5 - 5 hr . ) . Postgate
. & Hunter ( 1 9 6 2 ) quoted death rates in %/hr . since thei r
survival curves for starved A . a erogenes t ended to b e linear .
Survival curves for S . l acti s were generally sigmoid in shape ,
so that it i s probably more rea sonable to quote the death rate
a s the time taken to r each 5 0% vi ability .
In de-ioni zed water containing
Effect of salt concentration .
10� M-EDTA the viability of resuspended organi sms decreased to
5 0% in 3 . 6 hr .
In N a 2 H P0 4 -KH 2 P0 4 buff er ( pH 7 . 0 ) containing
10 � M-EDTA , survival times decrea sed slightly with increasing
buff er concentration .
Organi sms in phosphate buffer at
0 . 0 7 5M, ,O . 1 5 0M and 0 . 3 0 0M decreased to 5 0 % viability in 7 . 2 hr . ,
73·
6 . 1 hr . , and 6 . 0 h r . , respectively .
Ringer solution , with
and without 1 0� M-EDTA , gave 50% vi abiliti es after 6 . 7 hr . ,
and 3 . 8 hr .
All times were + 0 . 3 hr . for replicate samples .
Effect of pH value .
In pho sphate-citrat e buffer at 3 0 0 ,
S . lactis ML 3 had a sharp pH optimum fo r surviva l near 7 . 0
( Fig . lla ) .
Survi val times decreased sharply on d ecrea sing
the pH va lue and a 5 0 % decrease in viability occurred in
0 . 5 h r . at pH 4 . 0 .
It was therefore of interest to r e - examine
the obs ervations of Harvey ( 1 9 6 5 ) who concluded that S . lactis
ML 3 organi sms held in a broth growth mediUm adj ust ed to pH 4 . 2
wer e damaged bUD maintained complete viability for 5 hr .
O rgani sms were grown and placed in a broth medium adjusted to
pH 4 . 2 a s described by Harvey ( 1 9 6 5 ) .
It was found that
9 7 -9 9% of the o rganisms survived for at least 6 h r .
This
indicated a very ma rked protective eff ect of nutri ents at
low pH values .
O rgani sms harvested f rom Harvey ' s �growth
medium ( pH 6 0 5 ) and starved in pho sphate buffer gave simi l a r
sur vival times t o washed organisms f rom the routine medium
sta rved in pposphate buffer .
Since Dawes & Ribbons ( 1 9 6 2 , 1 9 6 4 ) have suggested that
an energy sour c e is r equi r ed for intracellular pH control
in starved bacteria it s eemed appropriate to r e - examine the
pH eff ect on S . lactis in the presence of an add ed energy
sourc e .
As addition of either carbohydrate or arginin e alone
produced acc elerated death of starved S . lactis organi sms ( s ee
F i g . 1 4 ) " it was decided to r e- examine the pH effect with
2
added Mg + , whi ch aboli shed arginine - accel erat ed d eath ( see
2+
F i g . l 4b ) , in addition to incubations with arginine + Mg .
2+
Addition of Mg
alone decrea s ed the d eath rate at all pH
values ( Fi g . l Ib ) , whi l e arginine + Mg 2+ produced a further
r emarkabl e decrease in the d eath rates ( Fig . llc ) .
Note the
different time scales in Fig . lla , b and c .
Results are
summari z ed in F i g . lId .
The initi al pH values in Fig . lla , b ,
did not change significantly o ver the starvation period .
After 4 8 hr . starvation with 10mM-arginine (Fig . llc ) , the
75
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(c)
+ Mg 2+
+ ar,;jininoZ
OL-----��----�12�--�18�--�2�4
TIME (trs)
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0L-------�15�--��--�4�5TIME (hrs)
Eff ect o f pH on surviva l of Streptococcus lactis
Fig . 1 1 .
ML 3 .
Buffers were prepa r ed from 0 . lM-N a 2 HP0 and 0 . 0 5M- citri c
4
Part ( a ) .
Bacteria wer e
acid containing 10� M - EDTA .
harvested f rom Z Oml . culture at the end of the growth pha s e ,
washed and r esuspended i n 5ml . pho sphate-citrate buffer ( pH 7 . 0 ) ,
0 . 5ml . of thi s suspension was added to 10ml . buffer at 3 0 0 to
give 1 9 0 ;u g . /ml . and final pH values (±0 . 0 5 ) of : €) , 7 . 9 3 ; ()
Part ( b ) .
6 . 9 8 ; 0 , 6 . 5 5 ; D , 6 . 0 0 ; � , 4 . 9 0 ; A. , 4 . 0 3 .
Su�pensions prepared a s in Part ( a ) at simi lar pH va lues .
Z+
Part ( c ) .
Suspensions
Each suspensipn contain ed 1 mM_Mg .
prepar ed a s in Part ( a ) but at 8 0;U g . dry wt . bact eri a/mI .
The
buffer contained 1 0mM- arginine and pH values were adj ust ed to
tho s e above by addition of HCl .
Part ( d ) .
Summary of results
from Parts a , b , and c .
Before vi abi lity determination samples
2+
were di lut ed in pho sphate - citrate buffer containing l m�-Mg
( pH 7 . 0 ) .
I
74 .
initial pH values o f 7 . 9 8 , 6 . 9 8 , 6 . 0 5 , 4 . 9 2 and 4 . 0 5 had
changed to 8 . 1 6 , 7 . 0 8 � 6 . 1 4 , 5 . 11 and 4 . 1 9 r espectively .
This was pr esumably due to ammoni a production .
Harvey ( 19 6 5 ) r eported that when growing S . l actis
ML 3 o rgani sms were subj ected to rapid pH changes from
4 . 2 - 4 . 7 to 5 . 2 - 6 . 3 , a lag resulted b efore the normal growth
Thi s lag did not occur when
rate for the new pH was as sumed .
the initial pH was 5 . 0 or above .
It was suggested that growth
b elow pH 5 . 0 resulted in some unspecified damage to the cells .
Growth was mea sured by turbidity increase .
Simi lar experiments
have now been repeat ed with additional measurement s o f total
Whi l e
chain counts , plate counts and slide-culture viability .
t h e turbidity increased b y about 5 0% i n 6 hr . a t p H 4 . 2 , there
wa s no signifi cant increase in total chain counts or plate
counts ( Fi g . 1 2 ) .
No aggregation of cells was evident so
pr esumably cell growth and di vi si on were no longer in a
steady state .
Synthesis o f important c ell components at
very di fferent rates is known to occur when certain nut ritional
or physio logi cal imbalances exist in a growth medium ( s ee
Duguid & Wi lkinson , 1 9 61 ; Shockman , 1 9 6 5 ; Bazi ll , 1 9 6 7 ;
E l sden , 1<»7 ) .
A ssuming that a mass increase occurred without
c ell divi sion at low pH , then t ransfer to a medium with a pH
allowing cell divi sion would be exp ected to produce a lag·
in the growth curve until the normal cell size di stribution was
resumed .
I f the growth curve i s extrapolated back to the
I normal turbi dity I correspond'i ng to the total cell numb ers at
z ero time , then the lag is partially eliminated . Further ,
the plots of total chain counts and plate counts do not show
corresponding lags .
Results of a typical experiment are
shown in Fig . 1 2 .
This may partially explain some o f the
r esults repo rted by Harvey ( 1 9 6 5 ) .
Effect of temperatur e .
The death rate decreased markedly on
lowering the incubation temperature of washed suspensions from
4 5 0 to 3 0 ( F i g . l 3 a , b ) .
N ote the time scale for Fig . l 3b is
t en times that fo r Fig . 1 3a .
pH
4·2
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TIM E
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2
4
Fig . 1 2 .
Eff ect o f growth pH on Str eptococcus lacti s ML 3 .
O rganisms were grown , harvested and subj ect ed to pH changes
by addition of fresh medium as described by Harvey ( 1 9 6 5 ) .
Measur ement s were made of bacterial ma ss , � ; total chain
count , [J
and plate count , () ; as d escribed in Methods .
Some o f the sampl e s were cooled and stor ed at 40 fo r short
interva l s b efo r e total count d eterminations .
Verti cal bars
r epresent standard d eviations from the mean , total counts were
determined in quadrupli cate and t en plates were count ed for
each : det ermination .
Slide - cultures ·indi cated 9 7 -9 9 % viability
throughout the experiment .
(a)
7i� 5 4 ·
5
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60
90
Fi g . 1 3 .
Effect o f temperature on survi val of Str eptoco ccus
Cell suspensions were prepared as for Fig . 8 ,
lactis ML 3 .
the washed c ells b eing inoculated into
equi librated to the sto rage incubation
°
°
°
� , 4 5 ; 0 , 37 ; 0 , 30 .
Part
°
°
°
22 ; �
16 ; � , 3 .
T emperatures
°
cultures were incubated at 3 0 .
,
phosphate buff er
t emperature .
Part ( a ) :
°
( b ) : O , 30 ; & , .
°
a r e + 1 , slide­
75 .
Effect o f atmo sphere and agitation .
Both air and commercial
' O Z - free l N Z inc r ea s ed the death rate when gently bubbled
through washed suspensions ( 5 0% decrea s e in viability in
4 . 5 - 5 hr . for air and in 3 - 3 . 5 hr . for N Z ) �
Addition of
sodium thioglycollate ( Z O mM ) to stati c suspensions did not
Toxi c H z O z accumulated when growth
affect the d eath rate .
cultures o f S . lactis o rgani sms were a erated ( Dr . G . R . Jago ,
pers . comm . ) .
However , it seems unlikely that suffi ci ent
H Z O Z would a ccumulate in starved suspensions , at low bacterial
aensiti es , t o increa s e the death rat e .
The results imply
that speci al E values wer e r equir ed for survival , as would
h
befit a mi cro-a erophil i c organi sm .
Gentle agitation produced by a magnetic stirrer had no
measurable effect on survi val curves compared with thos e of
stati c suspensions , but vigorous agitation ( magneti c stirrer )
generally gave more rapid death ( 50% decrease in vi abi lity
in Z - 3 h r,. ) .
Vigo rous agitation did not appear to produce
chain breakage but may have increa s ed the rate of l eakage of
solub l e intracellular components .
The agitation eff ect
appears to be distinct f rom the a eration eff ect .
Effect o f metabolic inhibitors .
The addition of O . O lmM­
iodoacetate decrea s ed survival times sharply ( Tabl e 8 ) .
Methylene blue at concentrati ons above O . O ZmM showed an
immedi ate bactericida l eff ect , whi l e at O . OOlmM the d eath
rate increased slightly .
The addition of Z . 7mM-sodium
arsenate and Z . 4mM-sodi um fluoride also produc ed a slight
increa s e in d eath rat e .
Sodium a zide , Z . 4-dinitrophenol ,
potassium cyanid e and sodium malonate did not signi fi cantly
influen c e the survival curves , a lthough all experiments wer e
Before slide - cultures
made under conditions o f low 0 z -tension .
were pr epared the inhibitors were removed by wa shing ( s ee
Methods ) , thi s pro c edur e had no noti ceable eff ect on the
vi ability of control populations .
76.
Table 8 .
Eff ect of metabo lic inhibito r s on survival o f
streptoco c cus 1actis ML in buffered suspensions at 3 0 0 .
3
O rgani sms were harvested at the end of the growth pha s e , wa shed
twi c e and resuspended in 0 . 07 5M-pho sphate buffer at equi v .
2 0�g . dry wt . organi sm/mI .
Buffer ( pH 7 . 0 , except where
indi cated ) contained 10/ M-:-EDTA and inhibitor a s below .
Concentration
I nhibitor
(mM)
None ( pH 7 . 0 )
None ( pH 6 . 5 )
2 , 4-Dinitroph eno1
I odoa c etic acid
b
Methyl en e b lue
Potassium cyanide
Sodium arsenate
SodiUJ!l azide
Sodium fluo ride
Sodium malonate
b
1 . 10
0 . 11
0 . 11
0 . 01
0 . 010
0 . 00 5
0 . 001
1 . 54
0 . 77
2 .70
0 . 54
1 . 54
0 . 15
2 . 38
0 . 24
6 . 75
0 . 67
Death rate
a
7·2
6.0
6.8
6. 6
1.1
1.9
2·9
5.0
6.7
7.0
6.8
6.1
6.8
6.8
7.5
6.9
7.3
6. 3
6.0
a -- T ime ( hr . ) for 5 0% decrease in viabi lity (± 0 . 3 hr . ) .
b
Buffer adj ust ed to pH 6 . 5 .
77 .
Effect of a dded carbohydrates .
and fructo s e ( 10 mM ;
Lacto s e , gluc o s e , galactos e
Fig . l 4a ) produced markedly increa s ed
death rates in washed suspensions ( 5 0% decrease in viability
in 1 - 1 . 5 hr . ) i r respective of the growth phas e o f the
organi sms and th e limiting nutri ent ( 5 0% decrea s e in vi ability
in 0 . 5 - 0 . 7 hr . for organi sms from amino acid-limit ed medium ) .
The suspension r emained at pH 7 . 0 and analys es for lactic acid
indi cated that f ermentation o f all carbohydrates took p�ac e .
Sodium lactate Uo mM ) had no eff ect on survival times .
Accel erated death was markedly reduc ed in all cases on additi on
2+
of 1 mM_Mg , giv ing survi val times similar to control systems
2+
without c arbohydrat e or Mg
( Fig . l4a ) .
Addition of
Analysi s for lactic acid showed
1 0 mM-ribose had no eff ect .
that S . lactis ML 3 did not f erment exo genous ribose to
lactate in washed suspensions .
A population density effect ,
similar to that shown in F i g . lOa , was obs erved in the
pres ence of added gluco s e ( 10 mM ) .
Effect of a dded amino acids and other growth medium component s .
Organisms resuspended in routine medium without lacto s e ,
vitamins and NaHC0 3 , survi ved for a longer period than organi sms
2+
r esuspended with Mg
only ( Fig . l4b ) .
In the former syst em
no signifi cant change in the total number of cocci occurr ed
up to 2 3 hr . in two suspensions containing 100� g . dry wt .
o rgani sm/mI . , one suspension contained p eni cillin G to prevent
These r esults
c ell growth and provide a control ( T ab l e 9 ) .
indi cate that cell divi sion or cryptic growth is unlikely to
o c cur in starved suspensions o f S . lacti s ML 3 .
2+
Casamino acids + Mg
gave equiva l ent survival as in
routine medium without lacto s e , vitamins and N aHC0 3 , whil e
2+
a rginine + Mg
was almost a s effective ( Fig . 14b ) . Arginine
2+
produced acc elerated d eath in the absence of add ed Mg ( Fig . 14b ) .
Alanine , a spartic acid and glutami c aci d , whi ch were subs equently
found to compri s e most of the free amino acid pool in these
organi sms , produced a ma rginal increa s e in survival time when
2+
incubated together with washed organi sms +Mg
Glycerol +
2+
Mg
a{so produc ed a marginal increa s e in survi val time
75
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TIME (hrs)
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9
15
30
TIME(hrs)
45
Fi g . 1 4 .
Effect o f nutri ents on survival of Strepto co ccus
lactis ML 3 .
Cell suspensions were prepared a s for Fig . 8
Part ( a ) : 0 ,
in pho sphate buffer containing 10�M-EDTA .
60
+ l acto s e ; � , + glucos e ; [] , + galacto s e ;
no additi on ; �
+ lacto s e + Mg 2+ ( 1 mM ) .
All carbohydrate supplements were
2
+
l OmM .
Part ( b ) : � , + Mg ; () , + Casamino acids ( Difco , 0 . 5% )
2+
+ Mg ; [J , + arginine ( lOmM ) + Mg 2+ ; X , + arginine ( lOmM ) ;
2+
+ alanine , a spart i c a ci d , glutami c aci d ( each 10mM ) + Mg ; � ,
2+
2+
growth
supplements lmM ) ;
+ glycerol ( lOmM ) + Mg ; ( all Mg
medium minus lacto s e , vitamins and N aHC0 3 .
,
T ab l e 9 .
C onstancy o f tota l c el l numb ers o f s t r ept o c o c cus l a c t i s ML
p r e s enc e o f amino a c i d s .
sta rved i n the
3
Susp ensions were p r ep a r ed as d escri b e d i n T a b l e 7 at appro x .
- lOO � g . dry wt . bacteria/mI .
Resusp ension buff ers c ontained 0 . 0 7 5M - pho sphat e ( pH 7 . 0 ) ,
10 � M - EDTA , ImM -MgS0 ; plus t h e f o llowing addition s ;
A , no a dditi o n ;
B , ca samino
4
acids ( Di f c o , 0 . 5% ) + arginin e ( lOmM ) ;
c,
as for B + p eni cil lin G ( 10 0 units/mI . ) .
Turbi dity and total c o cci were det ermined a s d es c ri b ed in Meth od s , standard devi ations
are for four d et erminations .
Additions to above buffer
Incubation
time ( hr . )
None ( A )
T urbidity
Amino a c i d s {B}
Turbidity
Total cocci
8
( x 10- )
Ami n o a c i d s
Turbidity
0 . 25
· 359
· 355
1.5
· 346
· 349
4.5
· 320
· 3 31
· 334
10
. 28 5
· 317
· 311
23
. 268
. 29 3
29
. 263
. 291
47
. 141
. 27 2
53
: 130
. 2 60
Note :
8 . 27
8 . 04
+
+
. 23
· 31
. 358
. 351
. 28 2
+
peni ci l l i n { C }
Total c o c ci
8
( x 10 - )
8 . 01
+
. 25
8 . 13
+
. 27
. 27 8
7 . 47 + . 4 4
. 253
7 · 39 + · 52
. 222
Mi c r o scopic examina t i o n r evea l ed some c el l l ysi s in a l l sus p en s i ons at 47 hr .
"-l
00
79 .
2+
whi l e 10 mM-sodium a c etate + 1 mM_Mg
was ineff ecti ve ( Fig . 1 4b ) .
Washed o rgani sms incubated with the vitamins , purines and
pyrimidines supplied at the routine medium concentrations were
2+
without eff ect in the presenc e of 1 mM-Mg .
Growth charact eri sti cs o f survi vors .
Survi ving organi sms
from some resuspended systems had considerably longer divi sion
times than exponentially growing o rgani sms .
These times wer e
measured ( a s shown in Fig . l 5b ) in four resuspended systems
giving the maximum scatter in survival times .
Divi sion lags
t ended t o increa s e j ust b efor e the onset o f bacteria l d eath ,
the increa s e b eing parti cularly sharp in systems without
2
Mg + ( Fi g . l 5 a ) .
This method gives only an approximate
division lag time and errors are likely to b e considerable
at low viabiliti es ( s ee Fig . 7 ) .
In most experiments where organisms were incubated in
2
o rganl. sms on s l 1· d e-cu It ures
. .
th e a b s ence 0 f M g + , survlvlng
showed a large proportion of morphologi cally aberrant colony
forms onc e the starved organisms started to di e ; instead of
the typi cal circular coloni es many organisms produc ed
elongated colonies and these organi sms generally� showed longer
division lags .
PART I I .
CHANGES IN VIABLE ORGANI SMS IN STARVATION CONDITIONS
i ) Chemi cal Studies
Organisms were harvest ed from the end of the
' Reserve polymers .
growth phas e in routine medium and extracted for polyglucose
and poly-�-hydroxybutyrate ( PH B ) a s describ ed in the Methods .
Subsequent ana lysi s o f the extracts r eveal ed no tra c e o f either
polymer .
However , the routine medium was lactos e-limiting so
that optimum conditions may not have exi sted fo r polymer
a ccumulation .
When organisms wer e harvested at the end of the
�rowth pha s e and resuspended in the routine medium minus the
casamino acids and p eptone components , no detectabl e synthesis
Conditions simi lar
o f either po lygluco s e o r PHB occurred .
(a)
•
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Fig . 1 5 .
Divi sion lag times o f survi ving Streptococcus la ctis
ML ' � rgani sms ln various resuspended syst ems .
Cell suspensions
3
were prepared as for Fig . 8 in pho sphate buff er containing
l0 M-EDTA . , P art ( a ) .
Vi ab i liti es for the suspensions :
, ;U
+ 0 . 5 % Casamino acids ( Difco ) + lmM-Mg 2+ ; + lmM-Mg 2+ ; no
addition ; + 10mM-l acto s e ; a r e indi c ated
/l.
x ,
,
respecti v ely .
Divi sion lags o f survi ving c ells ln the same
systems a r e indi cated 0
0 , /::.
X
Part ( b ) . Method
,
,
o f cal culation o f divi sion l a g time .
80 .
to the s e were found to be optimal for intracellular po lygluc o s e
synthesi s in Streptococcus salivarius ( Hamilton , 1 9 6 8 ) .
Therefore it seemed unlikely that S . lactis was capable o f
synthesi zing the s e polymers whi ch are found in many bacterial
speci es .
Electron mi crographs of thin s ections showed no
evidence of polypho sphate granul es .
Oxygen uptake studi es .
Spendlove , Wei s er & Harper ( 19 57 )
claimed that a strain o f S . lactis was capable o f ' active a erobi c
r espiration ' a n d that starved organi sms began t o ' oxidi z e some
It was suggested that thi s
endogenous substrate after a lag ' .
substrate wa s either lactate or succinate .
In vi ew of thi s
unconfirmed r eport i t wa s d ecided to mea sur e the o xygen uptake
The
of S . lacti s ML in the presence of various substrates .
3
oxygen uptake of o rgani sms starved in pho sphate buffer was
insignifi cant , and of the substrates added only gluconat e and
pyruvate produc ed significant oxygen. uptakes ( T able 1 0 ) although
other substrates may not have been taken up by the c ell s .
During the f ermentation of added carbohydrates , variable but
small amounts of oxygen .we� e taken up by the organi sms ( T able 11 ) .
The oxygen uptakes measur ed in the pr es ence and absence of
gluco s e wer e similar to thos e reported for S . diacetilactis
( Ob eFman , 1 9 6 2 ) .
Changes in bacterial protein and total N .
Only slight , i f
any , n et protein br eakdown o ccurred in starved suspensions
( Fi g . 1 6 ) .
Prot ein was e stimated by the biuret method , whi ch
mea sures peptide bonds and by the Folin-Ciocalteu procedure
whi ch measures tyro sine or indolyl residues ( s ee Methods ) .
Only traces o f free tyro sine wer e relea s ed from starved
organisms ( T able 1 3 ) .
Protein accounted for 4 8 % of the
initial bacteri a l dry wt . and starvation for the 28 hr . period
result ed in a 2 6% b acteri a l mass loss ( Fi g . 1 6 ) .
Soluble protein ( 0 . 2 2 mg . lml . ) , amounting to 10% o f the
total bacteri al prot ein , was released from starved organi sms
into the external medium ( Fig . 1 6 ) .
The total N in this
culture was constant at 0 . 4 9 + 0 . 01 8 mg . lml . and the amount
81 .
Table 1 0 .
starved
3
in pr esen c e of pot entia l substrates .
Each Warburg flask
contained 1 5 m� dry wt . washed bacteri a , 3 0 umol es substrate
/
and 1mM-MgS0 4 in 3 . 0 mI . O . 07 5M-pho sphate buff er ( pH 7 . 0 )
Substrat e
Acetate
Butyrate
Citrate
Glycerol
G1uconate
Lactate
Malate
Propionate
Pyruvate
Succinate
Oxygen uptake by streptococcus 1 a ctis ML
1 hr .
Total 0 2 -uptake � .
3 hr .
2 hr .
)
6 hr .
8
11
15
18
7
9
21
5
8
5
21
8
11
6
53
9
12
8
48
11
15
14 ,
14
8
82
12
14
11
65
14
7
8
6
33
9
22
25
15
99
20
16
19
83
23
82 .
Table 11 .
Oxygen uptake by streptococcus 1acti s ML sta rved
3
in the presence of carbohydrat es .
Each Warburg flask contained
5 mg . dry wt . wa sh ed organisms , 10 ;umo 1 es substrate ( exc ept
where speci fi ed ) and 1mM-MgS0 in 3 . 0 mI . 0 . 07 5M-pho sphate
4
buff er ( pH 7 . 0 ) .
Substrate
Gluco s e
G1uc1ose ( 3 0 ;t mo1 e )
Galactos e
L acto s e
F ructose
Maltos e
a
Arabinos e
a
Ribo s e
a
--
Total O -uptake (jd . )
2
1 0 min . 3 0 min . 6 0 min . 1 2 0 min . 2 4 0 min .
2
2
2
3
5
19
24
28
53
32
86
36
98
39
105
33
27
23
17
1
0
87
66
135
87
98
27
3
3
17 4
95
149
31
4
3
191
97
165
32
5
6
Substrate not fermented .
54
24
3
2
4·5
. 100
�I
-..:.
01
E
50
.J
�
a:
w
�
�
tr
o
2
Total protain
Z
W
�
Q.
o
10
TIME
(hr)
20
30
Changes in protein in starved Streptococcus lactis .
Bacteria were harvest ed from the end o f the growth pha s e , washed
and resuspended at 3 0 0 in pho sphate buffer ( O . 0 7 5M, pH 7 . 0 ,
containing lmM-MgS0 4 + lO � M-EDTA ) .
At the times indi cated ,
samp�es o f the suspension were r emoved and a portion immediately
Fig . 1 6 .
d eep-fro z en � ogether with supernatant samples whi ch wer e obtained
after c entrifugation and filtration .
Protein analys es were
perfo rmed on who l e suspension ( ) ) and supernatant samples ( )
V erti cal bars repr e s ent S . D . from mean for 4 det erminations .
.
At each sampling tim � bact eri al d ensity ( [] ) was determined by
both turbidity and dry wt . measurement , and viability ( )
by slide�culture .
o f N r eleased into the suspending buffer after 2 8 hr .
starvation ( 0 . 0 9 1 ± 0 . 01 6 mg . /ml . ) suggested that a consi derable
amount of non-protein N was involved ( protein rel ea s e accounted
for approx . 0 . 0 2 mg . N/ml . ) .
The turbi dity of supernatant
samples was always l ess than 0 . 00 5 Uniti al turbidity of the
who le suspension wa s 2 3 . 1 ) .
Prot ein r elease appeared to b e
r educ ed � about the same time as the death rate increased .
However , the rate and amount o f protein relea s ed wa s not
influenc ed by the presen c e of exogenous amino acids which
r educ ed the d eath rate ( Table 1 2 ) .
The protein r e l ea sed from
S . lactis organi sms after 1 8 . 5 , 2 3 and 42 hr . starvation in the
experimental syst ems ( 1 ) and ( 2 ) ( Table 1 2 ) was a ssayed for
proteina s e and tributyrina s e a ctivity ( see Methods ) .
no detectable a ctivity was found .
Rel ea s e o f amino a cids and ammoni a .
However ,
The intrac ellular amino
acid pool of S . lacti s accounted for mor e than 3% of the dry
wt . of freshly suspended S . la cti s organi sms and thi s P9 0 1 was
rapidly depleted on starvation ( Fig . 1 7 ) .
Ninhydrin-reactive
materia l appeared concurr ently in the ext ernal medium and there
wa s a n et inc r ea s e in the total free amino a cids on starvation .
A chromatographi c investigation o f the ninhydrin-reactive
material wa s und ertaken .
Organi sms were a lways thoroughly
wa shed to avoid carry over o f amino acids from the growth
medium .
By two -dimensional thin-layer and paper chromatography
the maj or components of the intrac ellular pool wer e t entatively
identified from standard R values as alanine , aspartic a c i d ,
F
glutami c acid , glycine a n d threonine .
T h e glutami c a c i d spot
Thes e observations on the composition
wa s the most int ens e .
o f the amino acid pool are simi lar to thos e reported by
Bottazzi ( 1 9 5 9 ) for S . lacti s and by Holden ( 1 9 6 2 a ) for various
species of lactic a cid ba cteri a .
However , it is well
establi shed that the amino acid pool composition o f an
organi sm may b e markedly influen c ed by the growth medium
compo sition .
There appeared to be a progr essive loss of
84.
Releas e of protein from starved streptococcus
Table 1 2 .
lacti s .
Suspensions wer e prepared a s for Fig . 1 6 at an initial
density of 4 . 5 mg . dry wt . /ml . but with organisms resusp ended
in ( 1 ) 0 . 07 5M-phosphate buff er + ImM-MgSO 4 + 10 � M-EDTA and
( 2 ) as for ( 1 ) + casamino a cids ( 0 . 5% , Di fco ) + arginine ( 0 . 1% ) .
Supernatant protein wa s pr ecipitated with 5% TeA , wa shed and
estimated by the method of Lowry et a l . ( 19 51 ) .
Starvation
period ( hr . )
Suspension { I }
Sup ernatant a
Viabi lity
protein
(%)
2.5
6
99
98
18 . 5
23
42
93
71
0
. 03
. 06
. 17
. 20
. 29
Suspension { 2 }
Supernatant
Vi abi lity
a
protein
(%)
99
99
97
48
a -- Expr ess ed a s mean mg . /ml . of 4 d et erminati ons .
. 02
. 06
. 17
. 21
. 28
I
0
�
0
z
2
�
�I
�!
21
�
�
......
28
100
70 � I
2·
'-" I
If)
If)
«
2
-1
«
c:::
w
IU
«
al
40
1
�------�7�--�1�4�--�2�1--�2�8-J
TIME
r
I-1
Q5
«
>
10
(hr)
Fig . 1 7 .
Release o f ninhydrin-reactiye mat eria l and ammonia
· from starved Streptococcus lactis organi sms . Bacteria wer e
ha rvest ed , resuspended and sampled at interva l � , a s described
for F ig . 1 6 .
Bacterial extracts were prepared and all sampl e s
were deep-fro zen a n d later analysed ( s ee Methods ) .
Intrac ellular
free amino acid and ammoni a are shown a s ()
[] ; supernatant
amino acid and ammoni a are shown a s
Verti cal bars
Bact eria l
represent S . D . from mean for 4 d eterminations .
ma ss ( � ) a n d vi abi lity ( � ) were determined a s described in
Methods .
85.
intracellular amino acids with a corresponding increa s e in the
supernatant amino a ci ds ( Fig . 1 8 ) , suggesting that l eakage of
the intracellular pool occurred on starvation .
L ater in the
investigati on , an amino acid a nalyser was availabl � . This
confirmed that the maj or components present and the total amounts
of amino acids in the poo l ( Table 1 3 ) were similar to tho s e
found previously from colorimetric and chromatographi c analyses .
The 7 3 % net increa s e in the total free amino a cids after
2 8 hr . starvation ( T able 1 3 ) suggested that , in addition to
leakage o f intrac ellular amino a cids , some protein d egradation
o ccurred .
Thi s increa se in amino acid concentrations would
correspond to a 5% breakdown of the tdal bact erial protein .
Previous colorimetric methods o f prot ein estimation wer e too
insensitive to estimate these small changes a ccurately ( see
Fig . 1 6 ) .
No low molecular weight p eptides were observed on
the amino a cid traces but it is po ssible that i f they were
present they may have been removed when the samples were
d eproteini z ed .
The total amount o f amino a cids obtained
from hydro lysed organisms ( Tabl e 1 3 ) was consi stent with earli er
analyses for bacterial protein .
Glutami c acid, alanine and aspartic acid mad e up 4 9 . 2% ,
2 2 . 0% and 1 2 . 8% o f the initi al amino acid pool .
The total
amount of free a spartic acid was sub stantia lly reduced on
starvation whil e the glutami c acid level showed a slight d ecreas e
( T able 1 3 ) .
There a r e n o reports o f the cataboli sm o f these
amino acids by S . l a cti s .
The total amount of free lysine
increased considerably on starvation whil e other amino a cids
increas ed by varying amounts .
The l evels o f amino acids in
the intrac ellular pool reflect ed the total amino acid
N either free
compo sition o f the organi sms ( T able 1 3 ) .
arginine nor ornithine were detected in any samples .
These
results would indicate that it is unlikely that starved
S . lactis could obtain substantial energy from the amino a ci d
poo l .
Walker & Forrest ( 1 9 6 4 ) c laimed that ' en ergy from the
I
.
'
Fig . 1 8 .
Thin-layer chromatograms of the intrac ellular amino
acid pool and supernatant samples of starved streptococcus
Bacteria were washed twic e and resuspended ( 17 mg .
lacti s .
dry wt . /ml . ) in buff er at 3 0 0 ( see Fig . 1 6 ) .
Samples were
r emoved at intervals and c entrifuged .
The packed c ells were
washed once , resuspended at the original density in deioniz ed ·
water and extracted , the supernatant buffer samples were
desalted ( see Methods ) .
Equal volumes of c el lular extracts
( C ) and supernatant samp l es ( S ) wer e pla c ed on T LC plates ( a )
Plates were developed in phenol-water ,
and ( b ) r espectively �
dri ed and sprayed with ninhydrin ( s ee Methods ) .
Subscripts
denote sampling times in hr . The standard sample contained
5�g . of each amino acid indicat ed .
\
\ '
(a)
\
I
L EUCI N E
VAL I N E
A LA N I N E
THREONI N E
GLY C I N E
GL UTAMI C A C I D
A S PA RT I C A C I D
ST D
(b)
L EU C I N E
VA L I N E
ALAN I N E
THREON I N E
G L Y CI N E
GLUT AMI C A C I D
A S PA RT I C A C I D
ST D
86 .
T able 1 3 .
Rel ea s e of amino acids from starved Streptococcus
lactis organi sms .
Bacteri a were harvest ed , wa shed twi c e and
resuspended at a c ell density of 15 mg . dry wt . /ml . in buffer
( Fig . 1 6 ) .
The intracellular amino acid pool wa s extra cted
with deioni z ed water at z ero time ( s ee Methods ) .
After 28 hr .
sta rvation at 3 0 0 the organi sms in the buffer suspension were
wa sh ed and extracted in dei oni z ed wat er .
The supernatant
buffer was d eproteini z ed by addition of TCA ( final conc . 5% ) .
Samples ( 0 . 5 mI . ) of the extract s , supernatant and hydro lysed
bacteria wer e analys ed with an amino acid analys er ( s ee Methods ) .
Total
Hydrolysed
bacteria
Pool
( 2 8 hr.) ( 0 hr . )
48 . 5
9.3
8.0
0 . 74
18 . 6
Amino acid
Intracellular
Pool
O . hr .
28 hr .
Lysine
0 . 95
0 . 38
Super­
natant
28 hr .
8.9
Histidine
Arginine
0 . 24
0 . 03
0 . 71
4 . 25
1 . 32
0 . 20
16 . 3
0 . 15
0 . 10
0 . 03
3 . 44
0 . 11
0 . 22
1 . 24
0 .70
2 . 24
0 . 32
11 . 6
1 . 27
5.2
15 · 1
0.85
2 . 34
0 . 35
15 . 0
1 . 38
5.5
16 . 4
0 . 21
0 . 21
0 . 25
1 . 37
0 . 25
0 . 81
1 . 40
0 � 57
0 . 71
2 . 18
1 . 42
0 . 25
0 . 84
1 . 45
0 . 57
0 . 71
2 . 43
5.83
5l . 3 6
Aspartic a ci d
Threonine
Serine
Glutami c acid
Proline
Glycine
Alanine
Cystine
Valine
Methionine
I so l eucine
L eucine
Tyrosine
Phenylalanine
Ammoni a
Total amino acid
1 . 05
7.3
0 . 19
0 . 33
0 . 05
0 . 09
0 . 35
0 . 17
0 . 35
0 . 17
33 . l4
0 . 05
0 . 03
0 . 05
a
b
5 7 . 31
67 . 1
24 . 9
22 . 5
88 . 2
19 · 0
28 . S
56 . 6
23 . 2
9.7
lS . l
32 · 3
13 · 3
16 . 4
11 . 5
c
495 . 2
Results are expressed a s;u g . amino acid or NH /mg . dry wt .
3
bacteria at 0 hr .
a -- plus 6 uni dentifi ed peaks whi ch compri s ed 3 . 5 % total pool .
b
plus 4 unidentifi ed peaks whi ch compri s ed < 1% total pool .
c -- plus 1 peak whi ch compri sed 3% total pool ( probably
hydroxylysine ) .
87 .
d egradation of amino a cid- containing materia l was r esponsible
for maint enanc e and organi zation ' of starved S . faecali s
organisms .
The pro c ess wa s not defined but di d not invo lve
catabolism of amino acids .
These autho rs also r eported that
the total carbon liberated by S . fa ecali s o rganisms , when
starved under ana erobic conditions , could b e completely a c counted
for by amino acid r elease .
The dichromate oxidation method used
by them for total carbon d etermination ( Halliwell , 1 9 60 ) i s not
quantitative for all amino acids .
Mo st of the maj or component s
of the amino a cid poo ls of lactic acid bacteria . --a lanine ,
a spartic acid , glutami c a cid and glycine ( Holden , 1 9 6 2 a ) --- a re
only parti ally oxidi z ed by thi s metho d ( Table 1 4 ) .
Therefore
it seems likely that other c arbon compounds were released .
No
indication was given by Forrest & Walker ( 19 6 3 ) o r Walker &
Forrest ( 1 9 64 ) conc erning the standard used for total carbon
determination .
Changes in nuc lei c a ci ds .
Preliminary exp eriments indic ated
that a substantial amount of material , with an absorption
maximum of 2 5 7 JP' wa s relea sed from starved S . lacti s organisms .
Thi s suggested nucl ei c acid br eakdown with the release o f
ultravi o l et ( u . v . ) -absorbing purine and pyrimidine fragments .
Results from mo r e detai led measurements a r e shown in Fig . 1 9 .
2+
In the absence of added Mg , bact eri a l RNA wa s broken
down at a rapid rate from the onset of starvation , the
degradation products b eing released into the suspending buffer .
Organi sms initia lly contained 20 . �% RNA whi ch was r educed
after 28 hr . starvation to 5 . 5% RNA ( calculated on the initial
2+
b acteri al dry wt . ) .
With added Mg , RNA wa s broken down only
after a considerable lag and the d eath rate wa s also reduc ed ;
the initi al RNA l evel of 2 0 . 7% dry wt . was r educ ed to 10 . 4%
2+
after 28 hr . ( Fig . 1 9 ) .
Without added Mg , most organi sms
wer e still vi abl e when 5 0 % of the c ellular RNA had been lost ,
although with added Mg 2+ most organisms were non-viable at thi s
point ( Fig . 1 9 ) .
In both suspensions loss of c ellular orcino l-rea ctive
88 .
T able 14 .
Di chromate-oxidation of o rganic materi a l s .
The
following compounds ( 5 0 0� g . ) were subj ected to the H 2 S0 4 K Cr 2 0 7 o xidation method for the determination of 100-7 0 0� g .
2
o rganic mat eri al ( Halliwel l , 1 9 6 0 ) .
Results wer e compared
with di chromate blanks and sulphite-reduced di chromate blanks .
Compound
L -alanine
L -a spartic acid
L-glutami c acid
glycine
L -valine
L -threonine
L - l eucine
L - s erine
L -lysine . HCl
D-glucos e
Na 2 S0 ( O . lml . , 2 0 % , w/v )
3
Di chromate r eduction ( % )
0.0
5.5
6.7
0.0
71 . 4
45 . 1
88 . 1
46 . 5
46 . 2
68 . 2
100 . 0
<
o
10 T 1 M
19 .
Fig .
at the
in
o
o
L--------1�
- - �0
O---�----�
30
E - -2
T I MI ( h r )
E (hr) 20
B r e a k d o wn
o rgan i sm s .
0::
I­
� 81
�, i
iLl I
l o g-pha s e ,
end of the
no
M e th o d s ,
S.D.
and
O . 7 3mg .
and
f ro z en ,
c el lu l a r
and
f rom t h e m e a n
d r y wt .
l O/ M- EDTA
+
)
c o nt a i ni ng :
Bacterial masses were
/m l .
r e s p e c t i v el y .
ext r a c t s w e r e p r ep a r ed
l a t er
for
( pH 7 . 0 ,
S amp l e s
i n d i c a t ed a n d i mm ed i a t e l y c en t r i fuged .
times
r em o v e d a t th e
Supernatant
l a cti s
f r om routi n e growth m e d i um
o
wa s h e d o n c e a n d r e su s p e n d e d at 3 0
1nu'l1 -MgSO 4 '
,
additi o n ;
initi a l ly 0 . 7 4
were
s t a r v e d S t r eDt o c o c cu s
B a c t e r i a w e r e h a r ve s t ed
0 . 0 7 5M - p h o s p h a t e buf f er
,
in
o f RNA
ana lys ed .
V e rt i c a l
4 d et e rm i n a t i o n s .
as
describ ed i n
b a r s r ep r e s en t
materia l was simi lar to RNA loss but wa s not balanc ed by a
corresponding increase in supernatant orcinol-rea ctive mat erial
( Fi g . 1 9 ) , suggesting that some of the ribo s e may have b een
cataboli z ed . However , pr eliminary experiments showed that lactic
acid wa s not produced from exogenous ribo s e and sinc e the orcino l
r eaction do es not estimate pyrimidine bound ribo s e , it i s not
possible to balance ribo s e concentrati ons .
supernatan t
The E
2 5 7�
va lues , when convert ed to equi val ent yeast RNA , corresponded
clos ely with the bacterial RNA loss , suggesting that no
metabolism of nuc l ei c acid b ases took plac e . In later experiments ,
therefore , mea sur ements of the release of u . v . -absorbing
materi al into the suspending buffer wer e used as a measure of
RNA breakdown aft er samples had been treated with TCA to r emove
u . v . -absorbing protein ( s ee Methods ) .
The identity of the u . v . -absorbing compounds released from
starved organisms wa s studi ed by TLC and u . v . spectro scopy ( see
Methods ) .
A suspension with 5 . 3 mg . dry wt . organi sms/mI . was
prepared in pho sphate buffer without Mg 2+ .
After 24 hr .
sta rvation at 3 0 0 the supernatant showed an extinction
co effi ci ent at 2 5 7 � equal to 2 1 . 0 .
Supernatant samples gave
four di stinct u . v . -absorbing bands on c ellulose thin-layer
chromatograms .
There were no detectable bands with � values
corr esponding to nuc l eotides .
When the bands were elut ed and
their u . v . spectra record ed , the extinction rati os were not
in good agreement with tho se published for bases and nuc leosides
( Dawson et al . , 1 9 5 9 ) suggesting that each band contained mo re
than one compound .
However , from � values and ab so rption
maxima , the maj o r components may have been uridine , cytidine ,
hypoxanthine and adenosine .
The rates of RNA breakdown ( r elea s e of E
� material )
257
and loss of viability were mea sur ed in the pr esence of sub strates
whi ch produced the maximum range of death rate and cell divi sion
lag time ( Fig . 2 0 ) .
Glucose-accelerated d eath wa s a ccompanied
Organi sms in phosphate buffer
by extremely rapid RNA breakdown .
Additions
a lso showed rapid r at es of death and RNA breakdown .
�===;���
1 0 0 ����
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__
__
__
__
__
__
__
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75
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en
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25
o
t­
Z
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I
*
I
I
j:{
"P
... ...
... ...
-
----
z
0::
W
CL
.:::>
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I
I
J
�
I
;{
I
;.
/
/
/
.... -
� -
-
rn
C\I
wI
-­
� -
---
----
0·3
0·2
0·1
10
T I M E ( hrs)
15
20
25
30
Fig . 2 0 .
RNA breakdown and death rate of starved str eptococcus
l acti s o rganisms .
Suspensions were prepared a s fo r Fig . 1 5
a t 0 . 0 9mg . dry wt . bacteria/ml . i n phosphate buffer containing
10 �M-EDTA .
Supernatant E
values for the suspensions :
2 5 7m�
2+
+ 0 . 5% c a samino ' acids ( Difco ) + 0 . 1% arginine + 1 mM_Mg ;
2+
+ 1 mM-Mg ; no additi on ; + 10mM-gluco s e ; are indi c ated by
dashed lines and the symbols 0 , 0 , 6 , X , respectively .
Viability curves o f o rganisms in the same systems have so lid
lines .
90 .
o f Mg
2+
suppressed RNA degradation and the death rate but the
pres ence of exogenous amino acids had only a slight eff ect on
RNA br eakdown althuugh they markedly r educ ed the death rate .
Hen c e there wa s no general correlation b etween loss of vi ability
and RNA breakdown .
No detectable change in bacterial DNA occurred in two
experiment s wher e organi sms were starved for 1 6 hr . at 0 . 2 4 mg .
dry wt . /ml . in phosphate buff er .
The DNA content of the
susp ensions r emained at 8 . 8 4 ± 0 . 1 7 �g . /ml . ( i . e . DNA compri s ed
3 . 7 % of the bacteri a l dry wt . ) .
Changes in carbohydrate .
Previ ously, S . lactis organi sms wer e
shown to contain no carbohydrate whi ch could b e extracted by the
methods appropri ate for polyglucos e .
Analys es for c ellula r
anthrone-po sitive materi al and reducing suga r indicated that
ther e wa s litt l e , if any , ca rbohydrate breakdown in - starved
S . lactis ( T able 1 5 ) .
Carbohydrate fermentation would be
expected to produc e predominantly lactic a cid .
The sma ll amount
of lactate produc ed ( Table 1 5 ) suggested that about 2% of the
total c ellular carbohydrate may have b e en f erment ed .
Most of the hexo s e in S . lactis i s likely to have a
structural role .
0 ther investigato rs working with other
organi sms ( Strange et al . , 1 9 61 ; Ribbons & Dawes , 1 9 6 3 ;
Dawes & Ribbons , 1 9 6 5 ; Burl ei gh & Dawes , 1 9 67 ) have shown that
structural carbohydrate is only slightly degraded , if at all ,
in starved bacteria .
Changes in lipids .
S . lactis ML , grown in the routine medium ,
3
contained 3 . 8 % lipid o n a dry wt . basi s ( Table 1 6 ) .
A n et
l o s s of 1 0% of thi s lipid occurred during a 5 5 hr . starvation
p eriod .
During thi s time the po lar lipid fraction decrea s ed
from 8 5% to 7 0% o f the total lipid and the neutral lipid fraction
increa s ed from 1 2 % to 3 0% of the tota l lipid ( T able 1 6 ) . spot
int ensities on H S0 -charred thin-layer chromatograms indi cated
2 4
a marked increase in the free fatty acid compon ent of the
Hydrolysis o f po lar lipid may
n eutral lipid on starvation .
Table 1 5 .
C e l lular ca rbohydrate in starved St r ept o c o c cus l a c t i s o rgani sms .
Ba cteria from t h e end o f the growth pha s e wer e wa shed and r esuspended in 0 . 0 7 5M - pho spha t e
buf f e r ( pH 7·. 0 , + l 0 ;t M- EDTA ) at 3 0 0 and a d ensity o f 1 . 0 mg . dry wt . b a c t e r i a /ml .
At int erva l s , sup ernatant samp l e s w e r e prepared and f ro z en .
Anth rone - r e a c t i v e
carbohydrate wa s determi ned dir ectly o n wa shed o rgani sms .
Reduci ng suga r wa s measur ed
in wa shed organi sms a f t er storage at _ 2 0 0 �n 2 . 5N -H S0 before hydro lysi s .
2
4
C e l lular carbohwdrat e
Anthrone +ve
Reducing
.
ab
ac
mat erlal
suga r
Supernatant
d
--aA n t h r o n e-+
v e---a-c-t
te
La
�g . /mL )
mat eri a l
Starvation
peri o d ( hr . )
Vi abi l i t y
0.2
98
77
107
0.2
0.6
1.2
97
77
102
0.6
0.7
2
95
74
105
0.5
3
95
75
1 09
0.7
0.9
1.2
4
96
74
106
1.0
1.0
5
93
72
106
0.9
1.6
10
68
73
103
0.9
1.8
22
12
74
102
0.8
1.9
(%)
a - Express ed a s mean � g . gluc o s e e qui v . /ml . o f f our d et ermi nati ons .
b
S.D. < + 2.9 .
c - S.D. <+ 2.2.
d
S . D . < + 0 . 08 .
\.0
f-'
92 .
explain th es e changes .
The polar lipid pho spho rus and nitrogen
values ob s erved ( T able 1 6 ) wer e simi lar to tho se recorded by
Ikawa ( 1 9 6 3 ) for a range of lactic acid bacteria .
Separation of the polar lipid. fra ction by T LC revea l ed
s everal components all containing pho sphorus exc ept fo r the one
near est the solvent front .
A sample of thi s component was
s eparated by preparative TLC and elut ed .
Subs equent hydrolysis
and paper chromatography ( see Methods ) reveal ed that thi s
component wa s a glycolipid containing glucos e .
Several spots
near the o rigin were ninhydrin-po sitive .
Analys i s of the fatty acids in the neutral and polar lipid
fractions from S . lactis reveal ed a compo sition very simi lar
to that pr eviously report ed by MacLeo d , J en s en , Gander &
Sampugna ( 1 9 6 2 ) .
Starvati on brought about changes in the fatty
acid compo sition of the two fractions ( T abl e 1 7 ) .
The r elative
amount o f hexadecanoic acid decrea s ed in the n eutral lipid
fraction whi l e t etradecanoic acid increa s ed .
The cyclopropane
a ci d , lactobacillic aci d , increased in both n eutral and polar
lipid fractions with a corr esponding decrea s e of its immediate
precur so r , ci s-va c c eni c a cid . Formation of cyclopropane acids
invo lves the transfer of a methyl ene group from S-adeno syl­
methionine a cross the double bond of the corresponding mono enoi c
Kni vett & Cull en ( 1 9 67 ) reported
a cid ( Liu & Hofmann , 1 9 6 2 ) .
that E . coli , in the post -exponential growth pha s e , showed
increa s es in cyclopropane fatty acids with decreases in the
corresponding mono en6 ± c a cids and simi lar changes have been
report ed with many ba cteri al speci es after growth has stopped
( s ee Kates , 1 9 64 ) .
In a recent report by Steinhauer , F l entge & Lechowi ck ( 1 9 67 ) ,
it wa s suggest ed that the lipid patterns of mi cro-organi sms ,
a s d etermined by ga s-liquid chromatography of the fatty a cid
esters , could b e used in identi fication and differentiati on of
bact eria .
The s e �uthors examined the fatty a cids obtained
from the lipids o f S . lacti s and r eported the ma j o r components
a s C 1 8 : 3 or C 2l ( S O % ) and C : 2 ( 2 0 % ) .
Di - and tri enoi c acids
18
93·
Table 1 6 .
lacti s .
Changes in lipid components o f sta rved Streptococcus
Bacteria were grown at 3 0 0 in 3 x 101 . fla sks
containing 101 . routine medium , agitation was provi ded by a
Growth was followed turbi dimetri c a l ly and
magnetic stir rer .
at the times indi cated from the end of growth , bacteria were
harvested using a Sorva ll continuous flow centrifuge .
With a
flow rate o f 61 . /hr . at 3 l , 0 0 0 g , turbi dity measur ements on the
supernatant i ndi cated recovery of :> 9 9 % bacterial dry wt .
The
pa cked bacteria were wa shed twi ce in di sti lled water and the
lipids extracted , wa shed and analys ed as described in Methods .
Time from end of
growth ( hr . )
Bacter i a l yi eld
( gm . dry wt . )
a
a
Total lipid ( gm . )
( % bacterial dry wt . )
N eutral lipid ( gm . ) a
( % total lipi d )
Polar lipid ( gm . )
0
24
55
6.72
5.73
4 . 97
0 . 256
0 . 244
0 . 2 28
( 3 . 81 )
( 4 . 25 )
( 4 . 59 )
0 . 032
0 . 036
0 . 070
( 12 . 5 )
a
( % total lipi d )
Lipid r ecovery ( % )
0 . 218
( 14 . 6 )
0 . 2 01
( 30 . 6 )
0 . 15 9
( 85 . 2 )
( 82 . 5)
( 69 . 7 )
97 . 7
97 · 1
100 . 3
Polar lipid P ( % )
2 . 37
2 . 28
1. 86
Polar lipid N ( % )
0.72
0 . 67
0 . 95
Viabi lity ( % )
99
a -- Mean o f two o r more batches .
96
32
94 .
Table 1 7 .
Changes in fatty acids in starved Streptococcus
The neutral and polar lipid fra ctions o f the lipid
lacti s .
extracts in Table 16 were hydrolys ed , the fatty acids isolated ,
est eri fied and finally identified and quantitatively mea sur ed
by ga s-liquid chromatography ( see Methods ) .
Results are the
means of four analyses and a r e expressed as % of the total
fatty acid ester in each sampl e .
F atty acid
( probable
i dentity )
Polar lipid
2 4 hr . 5 5 hr .
N eutra l lipid
0 hr . 24 hr . 5 5 hr .
0 hr .
1.1
1.3
0.9
0.1
0.0
0.2
0.5
0.6
o.S
0.4
1.1
1.3
S.7
10 . 3
13 · 9
13 . 7
17 . 4
14 . 9
16 : l
3.9
4.1
4.2
4.1
3·7
4.2
C1 6 : 0
34 · 7
26 . S
25 . 9
20. S
20 . 7
21 . 4
24 . 6
26 . S
21 . 0
30 . 2
20 . 3
lS . l
2.7
2.4
1.4
0.1
1.1
O.S
24 . S
27 . 6
31 . 7
30 . S
35 . 7
39 . 2
C
12 : 0
C1 �
4 1
C
14 . 0
C
C
a
lS : l
C18 : 0
c
19 : 0
V
- Cis-va c c enic a cid .
The V sign i s used to indi cate the presence o f a cyclopropane
ring in lactobacillic a cid .
a
95 .
had not previously been r epo rted in bacteri al lipids ( Kates, 1 9 64 )
and it has been claimed that ana erobi c or facultative organi sms
c annot synthesi z e such a cids ( Bloch , Baronowsky , Go ldfin e ,
Furthermo r e ,
L ennarz , Light , Norri s & Scheuerbrandt , 1 9 6 1 ) .
lactob a ci l l i c acid , whi ch h a s been found i n t�e lipids of a l l
l acti c acid bact eria pr eviously investigated ( Kates , 1 9 6 4 ) was
not found by Steinhauer et al . ( 1 9 67 ) .
The disagreement
b etween the r esults of St einhauer et al . ( 1 9 6 7 ) and tho se of
the present investigation , whi ch confi rmed the earlier findings
of MacLeod et al . ( 1 9 6 2 ) , suggests that some erroneous fatty
acid identifi cations have b een made by Steinhauer et a l . ( 1 9 67 ) .
A small amount o f vo lati l e fatty acid ( 0 . 6� eq . /mg . dry wt .
ba cteria/ 2 4 hr . ) was r el ea s ed into the suspending buff er
( 0 . 07 5M-phosphate + 1 mM-Mg S0 ) by starved S . lacti s organisms .
4
The sour c e o f this materi al ha s not b een defined .
I n starved ana erobi c o r facultati ve ba ct eri a it s eems
unlikely that fatty acids could provide appreciabl e amounts of
energy for , even i f there were a � -oxidation pathway for the
production of ac etyl-CoA , there is no tricarboxylic acid cycle
to effect its complete o xidation .
( i i ) Protein synthesi s in sta r ved str eptococcus lactis
Protein synthesi s, a s d etermin ed by the mea surement of
14
valine _ c incorporation into c ell protein , wa s dependent
on the presence o f an exogenous en ergy sourc e ( Table 2 0 ) ;
ther e appear ed to b e no endogenous energy source capable of
supporting protein synthesis . Exogenous gluco s e produc ed
approximately four times a s much protein synthesis as arginine .
I t was sub sequently shown that glucose fermentation by S . lactis
generated AT P at about 7 . 5 times the r ate for arginine
metabolism and thi s may a ccount for the diff erent rates of
protein synthesi s .
Uptake of valine _ 1 4 C by starved organi sms occurred without
an exogenous energy source but to a much l esser ext ent ( Table 1 9 ) .
14
Ret ention o f the ac cumulated T CA so lubl e valine _ C by starved
Table 18 .
Summary o f changes i n components o f St r ept o c o ccus l a c t i s o r gani sms s t a r ved
at 3 0 0 in 0 . 0 7 5M- pho sphat e buff e r ( pH 7 . 0 , 10 � M-EDTA , lmM -MgS0 ) .
4
( 1 ) Dep l et i o n of po lymer s and amino a ci d p o o l ( r esul t s a r e expr e s s ed a s % init i a l
bact eri a l dry wt . ) .
St arvati on
period ( hr. )
Prot ein
0
a
Amino a C l" d
pool
48
28
40 . 7
Loss
b
7.3
c
Rl�A
d
DNA
e
Carbof
hydra t e
Lipid
g
Total
Bacteri a l
a
d r y wt .
3·3
20 . 7
3·7
7 ·7
3.8
87 . 2
100
0.6
10 . 4
3.7
7.2
3.6
66 . 2
74
2.7
10 · 3
0.0
0.5
0.2
21 . 0
26
( 2 ) Format i o n of products ( r esults o f ana l y s e s on t h e susp ending buf f e r e xpr e s s ed
a s % i n i t i a l b a ct erial dry wt . ) .
Sta r vati on
peri o d ( hr . )
L a ctat e
28
0 . 21
f
NH
V o l a t i l e fatty a c i d
( a s a c eti c a ci d )
0 . 36
0 . 22
a - Fig . 1 6 .
b
3
c
Estima t e d from protein r el e a s e d
a
Fig . 1 9 .
e - Page 9 0 .
f
Table 1 5 ,
g
T a b l e 1 6 ( 2 4 hr . ) .
h
Page 9 5 ( 2 4 hr . ) .
( carbohydrat e
Amino a c i d
5 ·73
c
and i n c r ea s e in t o t a l f r e e amino a c i d s �
c - Table 1 3 .
d
h
anthrone
+
ve ) .
c
Table 1 9 .
Val ine-
14
�
C uptake by starve d Strept o c o c cus l a ctis .
0 . 6 4 mg . dry wt . jml . , were starve d , at 3 0
c ontaining l sP M-EDTA ,
va l ine ( 2 0 0�g . jml . ) .
first column .
Wa s he d organisms ,
0
in 0 . 0 7 5M-phosphate buf f e r ( pH 7 . 0 )
14
1mM-MgS0 , DL-va l ine - 1 - C ( . 2 5? c . jml . , � g . jml . ) and L4
Addition s or omi s s i ons from this b uffer a re give n in the
Samp l e s ( 2ml . ) we re remove d at inte r v a l s and tre ate d a s de s cribe d
in Methods .
Time (min . )
Additions t o above buffe r
N one
+ Glucose ( 1 0mM )
+ L-a rginine ( 1 0mM )
+ L-arginine ( 1 0mM ) + c a s a­
mino acids ( 2 . 7 8mg . jml . ) -L
-val ine
+
7
30
45
90
120
619
659
446
318
185
154
1513
1981
2227
3 144
3090
3 180
705
898
934
1 3 64
1381
1378
49 9
703
845
904
9 12
856
Gluc ose ( 1 0mM ) + chloramph­
e n i c o l ( 2 0 9Ug . jml . )
+ �-arginine ( 1 0mM )
+ chlora­
mphenicol ( 2 0 0�g . jml . )
a - Based on initial b a cte rial ma ss .
b
60
190
14
a
Va l in e - C uptake ( c . p . m . /mg. dry wt . b a cteria )
17
Equivalent t o approx . 0 . 5% total va l in e -
14
C.
1934
2224
981
1 209
b
�
"
14
C inco rporation i nto c el l protein o f starved st r ept o c o c cus l a c ti s .
14
Thi s exp eriment wa s performed at the same time a s the val i n e - C upta k e exper iment
Table 2 0 .
Va l i n e -
( T a b l e 1 9 ) using the same c ondition s .
Samp l e s ( 2 mI . ) w e r e r emo ved at interva l s and
14
va l i n e - C incorporation into c e l l protein d et ermin e d ( s e e Method s ) .
Additions to buffer
Time ( mi n . )
7
Va lin e -
None
+
+
+
+
+
14
17
30
45
60
90
120
19 0
C incorporation ( c . p . m . /mg . dry wt . c e l l s )
9
11
11
14
20
11
247
471
626
8 01
1080
1 1 37
L - a r ginine ( lOmM )
44
91
167
243
242
265
L - a rgi nine ( l OmM ) + ca samino a ci ds
( 2 . 7 8 mg . /ml . ) -L-va l i n e
61
115
228
319
331
340
Gluc o s e ( lOmM )
Gluco-s e ( l OmM ) + chlo ramph eni c o l
( 2 0 �g . /ml . )
L - a r gi nine ( lOmM )
( 2 0 �g . /ml . )
+
chlo ramph eni c o l
a -- B a s ed o n initi al b a c t e r i a l ma ss .
152
214
28
34
a
--0
00
99 .
organisms was dependent on the presence o f an exogenous ene rgy
s ource .
At the bacterial density use d in Tables 1 9 and 2 0 ,
1 0mM-glucose woul d be completely fe rmente d in approx . 2 hr . ( see
F i g . 4 ) , while 1 0mM-arginine would re quire about 7 hr . for complete
metabolism ( see Fig . 2 2 ) .
Net protein synthe sis appe are d t o stop when the glucose was
e xhauste d .
By c ontrast , net synthe sis stoppe d in the presence
o f e xce s s arginine , e ven with casamino acids pre sent ( Table 20 ) .
S . lactis ML organisms c ontaine d 2 3 �g . valine/mg . dry wt .
3
( Table 1 3 ) .
Assuming that all the val ine is pre sent in ce ll
prote in and that the prote in synthe sized in starve d organisms
containe d the above proportion of val ine , then the amount of
prote in synthe sis in starve d organi sms can be calculate d from
14
the rate s of val ine _ C incorporation .
With exogenous glucose
and arginine , prote in synthe sis amounte d t o about 2 % and 0 . 5% of
the total cell prote in in the first hour of starvation .
The
apparent ce ssation of protein synthe sis after a period of starv­
ation in the presence of arginine may have re sulted from a
balance being rea che d between prote in synthesis and de gra dation .
Howe ver , no relevant data was available t o estimate the rate of
p rotein de gra dation in the presence of exogenous arginine .
Any
TCA-ins oluble prote in released from starve d organisms into the
external me dium during the se experiments would be measure d as
cell prote in .
Chloramphenicol cause d 7 8 % and 8 6 % inhibitions o f prote in
synthe sis in t he presence o f glucose and arginine re spe ctive ly
( Table 2 0 ) .
Chloramphenicol produce d l ittle or no inhibition
14
o f valine _ C uptake into the TCA s oluble pool ( Table 1 9 ) .
( Fo r the se cal culation s , re sults from Table s 1 9 and 2 0 were
graphe d to obtain the 4 5 and 90 �in . value s ) .
This is consistent
with the reports that chloramphenicol doe s not inhibit the
t ransport or a ccumulation of glutamate by micro-organisms
( Gale and Paine 1 9 5 1 ; Holden , 1 9 6 5 ) .
The ability of starve d organi sms t o take up valine _ 1 4 C
and to synthe s i ze protein appeared to be correlate d with survival
( Fig . 2 1 ) .
lob .
Protein synthesis wa s pr esumably influenced by RNA
degradation ( s ee F i g . 1 9 ) .
With the bacterial conc entration used
in F ig . 2 l , lOmM-arginine would b e metaboli zed in approx . 4 hr . (see
F i g . 2 2 ) and cons equently survi val wa s not enhanced to the same
extent as at lower b a ct erial densiti es .
I t has b een suggest ed that vegetative ba cteria may be able to
undergo ' adaptatio n ' in starvation conditi ons , pr esumably through
turnov er mechanisms , and that thi s may favour survi va l ( Willett s ,
1 9 67 i see also Clifton 1 9 6 6 ) . This could explain the decrea s ed
d eath rate o f S . l actis starved in the pres en c e o f arginine or
casamino a ci ds where some prot ei n synthesis took pla c e . I n fact ,
however , chlorampheni col app ear ed to r educ e the death rate in
some syst ems ( Table 2 1 ) . As well a s inhibiting protein synthesis ,
chlorampheni col may a lso inhibit prot ein degradation (Willetts ,
1 9 67 ) and h en c e may exert some sparing a ction on proteins essential
for survi val .
( iii ) Metabo li sm o f a rginine and gluco s e
A rginine wa s metabo l i z ed at a linear r a t e o f 2 . 3 5 � moles/mg .
dry wt . bact eria/hr . ( Fi g . 2 2 ) . This rate theoreti cally corresponds
to 2 . 3 5;u mol es ATP/mg . dry wt . bacteria/hr . ( ba s ed on the
stoi chiometric r eaction :
Arginine + H 2 0 + P + ADP � ornithine + 2NH 3 + CO + AT P ) .
2
i
The glycolyt i c activity of S . lactis ML 3 wa s O . 2 9 5� mo les lactate/
mg . dry wt . b a cteri a/min . ( F ig . 4 ) , and sin c e anaerobi c fermentation
o f gluco s e pro duces 1 mo l e of ATP/mo le l a ctate , thi s glycolytic
activity r epres ent s the formation of l 7 . 7 ;u moles ATP/mg . dry wt .
bacteri a/hr .
Therefo re the theor etical rate of AT P generation by
S . l actis i s a bout 7 . 5 times greater in the presence o f 10mM­
gluc o s e than i n the presence of 1 0mM-a rginine .
Arginine metabo lism yi elded the theor etical amount of ornithine
and slightly l es s than the theor etical amount of ammonia ( Fig . 2 2 )
confirming the findings o f Kor z enovsky & Werkman ( 1 9 5 4 ) . In earli e r
survi val experiments , washed suspensions with 2 0 "g . dry wt .
/
bact eria/mI . were i ncubated with 10mM-arginine . Assuming the above
linear rat e , these organi sms would requi re mor e than 2 0 0 hr . for
complete a rginine metabolism .
A glyco lyti c a ctivity of O . 2 9 5� mo les lactate/mg . dry wt.
bact eria/min . co rr esponds to f ermentation of 1 . 5 9 mg . glucos e/m g .
4
- -0. <l....
'0...'
. ,- '"
0
z 11 3
100
- -- -- -
•
Whole cel ls,
"
"
"
'
.......
,
O "-.!.
- cu
r: ' -
<t: l..
0:: c:Y
0
0
o.... CU
0:: .0
O +>
U �
?; >; 2
U
'<;f
......
'\.
...... '
'\.
'\.
"
,
'\
'\
'\
'\.
'\
'\.
75
'\.
,
\
-tJ1
EI
..... Ol
1
w -..,...:
� I
�
, a
E
..J 0
.....
x
'\
\
'\
\
\
'\
\
'0.
\
..-....
�
�
>f-
'\
\
\
50 ::!
ill
\
\
\
<:r:
:>
\
\
25
�I
0
o
5
TIME
(hr)
Fig . . 2l .
Ability of sta rved Streptococcus lactis to assimi late
14
and incorporate valine _ C .
Washed organisms were resuspended
at a d ensity of 0 . 9 3 mg . dry wt . /ml . in 0 . 0 7 5M-pho sph�te buff er
( pH 7 . 0 , + 10 � M-EDTA + lmM-MgS0 ) containing : L- arginine
4
( lOmM ) , ( 0 , ) ; no addition ( 0 , E] ) .
The cultures were
starved at 3 0 0 , sampl e s ( 2 ml . ) were withdrawn at intervals and
added to 2 ml . 0 . 0 7 5 M-pho sphate buffer containing MgS0 ( lmM ) ,
4
D-glucose ( 2 0mM ) , DL -valine-1- 1 4 C ( . 5 ;uc . /ml . , 1 2 ;u g . /ml . ) and
A ft er incub ation for I hr . at 3 0 0 ,
L -valine ( 4 0 0 ;u g . /ml . ) .
the ' inco rpo ration of valine - 1 4 C into c ells ( clo sed symbols )
arid protein " ( o pen symb o l s ) wa s determined ( see Methods ) .
' Re sults a r e b a sed on the initi al c ell mass .
The dashed lines
indi c at e slide-culture vi abilities .
EfiBct o f chloramph eni c o l on the survi va l o f str epto c o c cus l a c t i s .
Wa shed
organi sms , 3 0 � g . dry wt . lml . were sta rved at 3 0 0 in phosphat e buf f e r ( 0 . 0 7 5 M,
pH 7 . 0 , + 1 0 � M- EDTA + lmM-MgS0 ) containing the additi ons gi ven in the f i r st c o lumn .
4
Samples w e r e remo ved at interva l s and the o r gani sms wa sh ed ( exc ept wh e r e i n di c a t ed ) .
T ab l e 2 1 .
Vi ability was det ermined by the s l i d e - cultur e metho d .
Time ( hr . )
a
16
20
24
V i a b i l i ty ( % )
28
40
45
79
37
1
0
91
92
80
6
1
69
43
26
14
15
3
0
97
87
78
82
26
1
99
99
96
98
94
85
52
0
98
99
99
87
76
31
0
O
None
99
99
96
81
+ Chloramph eni co l (2 0 �g . Iml.)
99
0
98
99
99
+ Gluco s e ( l OmM ) + chlo ramph eni c o l (2 0 � g . Iml.) 9 9
99
+ Arginine ( l OmM )
chlo rampheni c o l (2 0 � g . Iml.) 9 9
Additions t o pho sphate buf f e r
+
+
Gluco s e ( lOmM )
A rgini n e (l OmM )
+
a -- Sli d e -cultur es pr epa r ed with unwa shed organi sms .
I-'
0
f--J
�
10t�------�--� 20
�E
�
8r
0
16
0
-3 6
12
r
i 4�
I
I
z
(!)
�-3I
<{
-
8
0:: I
o
w
z
E\
'-..:.
6
L
2:
<t
I
!
2-
4
0::
<{
o
20
TIME
( m i n)
40
60
A rginine metabolism by Streptococcus l actis .
Fig . 2 2 .
Bacteria were harvest ed from the routine medium at the end of the
growth pha s e , washed twi ce and resuspended in 0 . 0 7 SM-pho sphate .
buffer ( pH 7 . 0 ; containing lO �M- EDTA , ImM-MgS0 and 10mM­
4
L - arginine ) .
The organisms were incubated at 3 0 0 at a density
of 4 . 9 mg . dry wt . /ml .
At interva l s , samples were withdrawn ,
0
immedi ately coo l ed to _10 , c entri fuged and the supernatants
o
filtered .
Supernatant sampl es were fro z en ( - 2 0 ) and later
analysed fo r ·- a rgini ne ( 0 ) , ornithine ( 0 ) and ammoni a ( 6. ) ,
using the basic co lumn of an amino acid analyser ( see Methods ) .
dry wt . bacteria/hr .
102 .
Even a llowing for energy yield di fferences
from glucose metabolism , thi s rate was much greater than the
maintenanc e energy rate of 0 . 0 2 8 mg . gluco se/mg . dry wt . bacterial
hr . reported for E . coli by Marr et al . ( 1 9 6 3 ) .
As suming a simi lar
maint enance energy rate for s . lacti s , then clearly there i s no
economy of metabolic energy for maint enance of starved s . lactis
and the linear rate of glucose fermentation ( Fi g . 4 ) indi cates that
the metabolism of starved o rganisms continues at rapid rates .
O rganisms were starved in conditions gi ving the maximum r ange
of death rate and thei r glycolytic activity determined .
No general
correlation between glycolytic activity and survi val was found
( T able 2 2 ) . I t was shown that the incubation of starved organisms
with gluco se caus ed a mor e rapid loss of glyco lytic activity
( T able 2 2 ) . With the bact eri a l concentration used , 10mM-glucos e is
metabol i z ed in 5 . 2 hr . , and 1 0mM - a rginine in about 20 hr .
( iv ) Changes in permeability and ultra structure
Mea sur ement of bact erial lysis,l Leakage of metabolic intermediates
from viable S . lact i s organi sms was shown to occur from the onset
o f starvation . The relea s e of an intrac ellula r en zym'e , such a s
� - galactosida s e , into the ext ernal medium h a s been used a s an
index of cell lysi s ( Pollock , 1 9 61 ; Wi l l ett s , 1 9 67 ) . In the present
study no det ectable � -ga l a ctosidase activity was found in cell - free
samples of S . l actis susp ensions starved at a ba ct eri al density of
4 . 8 mg . dry wt . /ml . Citti et a l . ( 1 9 6 5 ) repo rt ed that the
(3 - ga lactosidase released from five oldl' of six strains of S . la cti s
by sho ck treatment was very unstable , so that i f thi s enzyme had
b een relea sed in the present study its a ctivity may have been
rapi dly lo st .
Assays for l actic dehydrogenase ( LDH ) indicated that thi s enzyme
wa s relea s ed from viable o r ga n i sms ( Table 2 3 ) . Rel ease of LDH
Control exp eriments showed
increa s ed at the onset of c ell death .
that the LDH in c ell - free samples wa s unstable under the incubation
conditions us ed .
Assays o f 2 4 hr . samples ( T able 2 3 ) , after 5 hr .
and 2 2 hr . incubation , indi cat ed a loss of 11% and 5 3% o f the
o ri ginal a ctivity .
Therefore , the results for LDH releas e in
T abl e 2 3 are only approximate .
Rel e� se o f ' diphenylamine reactive materia l ( exp� essed a s DNA T able 2 3 ) from sta rved o rganisms b ecame pronounced a s the death
rate accelerated .
Organi sms wer e washed
Glyco lyt i c activity o f starved St r ept o c o c cus 1 a c ti s .
and r esuspen d ed at 3 0 0 in 100 mI . 0 . 0 7 5M-pho sphat e buf f e r ( pH 7 . 0 , 1 0�M-EDTA ) containing
Table 2 2 .
the additions gi ven in the fi rst co lumn .
The c e l l ma s s wa s 0 . 2 1 5 mg . dry wt . /m1 .
At
lnt erva 1 s , samp1 es ( 1 0 mI . ) wer e r emoved and c entri fuged , the organi sms were washed and
r esuspended in phosphat e buff er ( 2 m I . ) and the glyc o lyt i c a cti vity det ermined as
d e scribed in Methods .
T im e ( hr . )
Additions to a b o v e buf f er
0.5
1·5
3
-5
10
22
27
. 17 4
(15)
. 155
( 1)
. 133
8
Glyco lyti c a ct i vi ty
None
+
MgSO
+ ' -MgSO
. 274
b
( 99 )
4
4
0 6 20
. 194
( 96 )
. 17 5
( 57 )
(1mM)
. 279
( 99 )
. 2 64
. 234
. 206
( 99 )
. 18 5
(96)
. 164
( 98 )
. 158
(72)
. 155
( 36 )
o..mM) + L - arginine
( 10mM)
. 285
( 99 )
. 2 69
. 262
. 21 8
. 18 9
. 19 3
( 97 )
. 14 8
( 98 )
. 14 4
( 91 )
. 286
( 99 )
. 27 2
. 241
. 231
. 17 8
. 17 0
(98 )
. 157
( 95 )
. 140
( 87 )
. 2 69
( 99 )
. 24 3
(72)
. 181
( 19 )
. 162
( 11 )
. 137
( 1)
. 11 8
. 052
. 045
. 277
( 99 )
. 260
. 20 5
. 17 1
( 99 )
. 121
( 84)
. 091
( 61 )
. 01 6
(23)
. 01 1
( 4)
+
MgSO Ltc (1mM) + L - a rginine
10mM) + c a samino a c i ds (. 5 %)
+
Gluc o s e (10mM)
+
MgSO
4
. 247
a
(1mM) + gluco s e (1 0mM)
a -- jUmo 1 es l a ct ate/mg . dry wt . b a c t eri a/min .
b -- % vi ab i l i t i es in bra cket s .
i-'
0
w
104 .
Table 2 3 .
Rel ease o f la cti c dehydrogenase ( LDH ) and
deoxyribonuclei c a c i d ( DNA ) from starved strepto coccus lacti s .
Washed organi sms were resuspended at 4 . 6 mg . dry wt . /ml . in
Samples
pho sphate buff er containing l0 ;UM-EDTA + lmM-MgS0 4 .
were removed at the times indi cated , c entri fuged and the
supernatant buffers a s sayed as described in Methods .
Starvation
Period ( hr . )
Supernatant
DNA
LDH
( Units/ml . )
yg . Jml . )
Vi abi lity
(%)
1
. 000
0.0
3
5
8
. 00 2
. 00 6
0.0
0.2
99
. 011
0.2
99
10
12
21
. 069
. 138
. 31 9
0.7
1.3
4.0
94
32
24
29
. 505
. 5 47
6.8
a - Equi val ent 6 . 6% total c ell DNA .
11 . 2
a
17
3
105 .
Eff ect of spermine .
C ertain a liphatic diamines , especially
spermine , have a marked stabilizing eff ect on osmotically
s ensitive bacteria and protoplasts ( Mager , 1 9 5 9 ; T abo r , 1 9 6 2 ) ,
suppressing the l eakage o f u . v . -absorbing mat eri a l and cell lysi s .
Incubation o f starved S . la cti s organisms with spermine r educ ed
the death rate and the relea s e o f u . v . -abso rbing materi a l ( Fig . 2 3 ) .
2+
This effect was l ess ma rked in the presenc e of Mg .
General ultra-structural f eatures of the cell .
Typi cal examples
of dividing cocci are shown in Plate 1 . This s equence i l lustrates
s epa ration o f the nucl ea r mat eri al . followed by s eptum formation .
Prominent meso somes ( Fit z-James , 1 9 6 0 ) are associated with , the
ingrowing c ell wal l and membrane and also with the nuc l ear
material .
The structure and possible functions of bact eri a l
meso somes have been r evi ewed b y Salton ( 1 9 67 ) and Ryter ( 1 9 6 8 ) .
It
has been suggested that meso somes may ( 1 ) function as sit es fo r
el ectron transport systems simi lar to mitochondri a , ( 2 ) have a
role in chromo some replication , ( 3 ) provide a mechani sm fo r the
secretion of extracellular enzymes and ( 4 ) have a r o l e in the
formation of new cell wal l and membrane materi a l thereby playing
an active ro l e in c ell divi sion . Some of these functions have been
questioned since mesosomes a r e rarely present in Gram-negative bacteria.
The cytoplasmic membrane i s 7 0- 8 0A thi ck and consists of two
prominent el ectron-dens e layers separated by a light l ayer ( Plate 1 ).
The cell wal l occurs as a diffus e band approximately 2 0 0- 2 5 0A
thick .
Cole ( 19 6 5 ) revi ewed investigations on c el l wall
r epli cation using an immuno fluo r escenc e t echnique and r eport ed
that cell wa ll growth in Str eptococcus pyogenes was initiated
along an equato ri al ring .
Cross wal l grew c entripetally with
the peripheral wal l r epli cating simultaneously so that the new
hemi spheres of the daught er cocci wer e initiated ba ck-to-back .
N ew equato rial sites of wa l l and cro s s wa ll fo rmation were initiated
befo re completion o f the pr evi ous cross wa l l . In contrast , Chung ,
Hawi rko & I saac ( 1 9 6 4 ) found that S . faecalis had only one site of
cell wall synthesi s per coccus . This mode of cell wal l
r epli cation app ears t o operate i n S . lacti s since only one
site of cro ss wa ll formation per coccus was s een ( see
Plate 2 ,
Fig . 1 ) .
Indi vidual cocci in n egatively stained
____. 1 75
�I
>- i
f�
cD
<[
>
50
25
- --
Fig . 2 3 .
Effect o f spermine on starved Streptoco ccus la ctis .
Or'g ani sms were washed and r esuspended at 2 0 ? g . dry wt . Iml . in
Viabi l iti es of
O . 0 7 5M-pho sphate buffer ( pH 7 . 0 , I O �M-EDTA ) .
suspensions containing : no addition , 0 ; spermine ( ImM) ,
( ImM ) , 0 ;
MgS0 ( ImM ) +
4
4
shown .
Release of u . v . -absorbing
dashed lines ; symb o l s as above .
Note : spermine Hel wa s steri li z ed
spermine conc entrations of 5mM and
depo sits in phosphate buffer .
MgS0
spermine ( ImM ) ,
, are
materi al is indi cated by
separately in disti l l ed wat e� ,
above produced crystaline
106 .
preparations o f whol e c ells wer e O . 5-0 . � wi de and O . 6-l . � long ,
depending on the stage o f di vi sion .
The fixation method of Kellenberger et al . ( 1 9 5 8 ) preserved
the nuclear mat erial with a typical fibri llar . structur e ( Plate 1 ) .
Bacterial nu�lei do not appear to be s eparated from the cytoplasm
by a membrane and may b e r egarded as a very long fi l ament of a
. singl e , tightly coi l ed DNA mol ecule ( Ryter , 1 9 6 8 ) .
Other
fixation pro cedur es c l early demonstrated granular parti cles ,
lOO - 2 0 0A ln di ameter , which were sometimes concentrated near the
cytoplasmi c membrane ( Plate 5 ) .
Most workers consider that the s e
parti cles are ribosomes ( see Kell enberger & Ryter , 1 9 64 ;
Silva , 1 9 67) .
.' • •
...;.'�1,
Although the appearance
Ultra structural changes in starved c ells .
o f ribo somes in thin s ections was neither uniform no r constant ,
examination of a large number o f s ections prepared by differ ent
techniques seemed to indicate a rapid d epl etion of ribosomes when
2+
organi sms were starved in buffer without Mg
( Pl ates 2 , 3 ) .
Magnesium starvation o f E . coli ( Morgan , Ros enkranz , Chan & Ro s e ,
1 9 6 6 ) and A . aerogenes ( Kennell & Kotoula s , 1 9 6 7 ) r esulted in
similar ribosome depl etion .
Death of S . l acti s in buffer without
2+
Mg
o ccurred whi l e cell structures r emained intact .
However ,
after 1 7 hr . starvation , intrusions of the membrane and cell wa ll
were obs erved whi ch appeared to be in di r ect conta ct with the
nucl ear material ( Plate 3 , Figs . 2- 4 ) .
These mi crographs are
typi cal o f organi sms whi ch have extruded their meso somes
( s ee Ryter & L andman , 1 9 6 4 ; Beaton , 1 9 6 8 ; Ryter , 1 9 6 8 ) .
Ryt er ( 1 9 6 8 ) r ep orted that meso some extrusion may occur without
loss of viabi lity .
After 3 0 hr . starvation in buff er containing Mg 2+ , the
viabi lity had fa1len to 42% and examination of mi crographs
indi cated that approximately 1 2% of the population had lys ed .
By 4 0 hr . most of the organi sms had lysed and the viability
was � 1 % ( Plate 4 ) .
Lysis appeared to r esult from rupture
of both the membrane and cell wal l .
Aft er lysi s , membran �s
t ended to r emain relatively intact whi l e cell walls fragmented
I
( Fi g . 1 , Plate 4 ) .
I t is not clear whether the sites involved
107 .
in the extrusion o f cytoplasmi c material were randomly ,
di stributed over the c el l surfac e , although in some cases they
appeared where meso somes would be expected .
The mi crogr aphs o f
lysing cocci ( Plate 4 ) appear similar to tho s e o f lipase-treat ed
bact eria ( Gho sh & Murray , 1 9 6 7 ) where b reakdown of the cytoplasmic
membrane and c ell wal l eventually r esulted in the liberation o f
cytoplasmi c materia l .
Autolytic enzymes a r e present in most bacteria and appear
to be a ctivated whenever normal growth o f the o rganism i s
di srupted ( see r evi ews b y Strip & Starr , 1 9 6 5 ; Shockman , 1 9 6 5 ) .
The bacterial c ell wall i s b eli eved to have a p redominantly
mechanical rol e , conferring cell shape , rigi dity , and r esistanc e
to the high o smoti c pr essure c r eated b y the s electivity of the
cytoplasmi c membrane .
As cocci lys ed , the cytoplasmi c membrane
pulled away from the rigid wall and eventually collapsed
( Plate 4 ) due to the decr easing o smotic p ressur e .
The membr anes
of Gram - positi ve bact eria are not bound to the c el l wa lls
( Salton , 1 9 67 ) .
I t i s interesting to note that whil e the c ell
walls of most Gram -positive bacteria a r e compri s ed mainly o f
mucopeptides ( up to 5 0 % dry wt . ) and t ei choic acids
( 30 - 4 0% dry wt . ) , O ram & Reiter ( 19 6 5 ) r eported that t ei choic
a ci ds were abs ent f rom the c ell walls o f group N strepto cocci .
Exogenous amino acids maintained c ell structures intact
A den s e ribo some pattern was
for a longer p eriod ( Plate 5 ) .
still vi sible after prolonged sta rvation and lysed organisms
appea r ed as the d eath rate increased .
EXPLANATI ON OF PLATES
Organi sms were fixed as described in Methods , exc ept wher e
indi cated . The bar represents O . 2 � on all micrographs and the
magnifi cation is x 6 4 , 4 0 0 except for Plate 1
Fig . 4 , Plate 5 ( x 21 , 00 0 ) .
(x
1 1 6 , 0 0 0 ) and
PLATE 1 .
Di vi sion s equence of Streptococcus lacti s ML from the
3
logarithmi c growth pha s e . Organi sms were fixed by the method of
Kellenb erger et a l . ( 1 9 5 8 ) which gives best definition of the
nuclear materi al and membranes .
Initial stage of cell divi sion showing the undivi ded
Fig . 1 .
nuclea r materia l ( N ) and sma ll invaginations where the mesosome
( M ) is continuous with the cytoplasmi c membrane ( CM ) . The
meso some a�pea r s to contain vesicles ( V ) .
Fig . 2 .
materia l
An int ermediate stage showing the divided nucl ear
( N ) in close contact with the meso some ( M ) .
Fig . 3 .
Cross wall formation i s complete with a clearly
defined membrane . The new mesosome ( M ) , formed at the next
divi sion sit e , i s continuous with the cytoplasmi c membrane .
The mesosomes in F i gs . 2 , 3 are of the lamellar type consi sting
of concentri c whorls of membrane , the appearance of meso somes
is partly dependent on the orientation of the section .
PLAT E
N
1.
PLATE 2 .
Figs . 1 - 3 .
Organi sms from the loga rithmi c growth
phas e .
Figs . 4 , 5 .
Organisms aft er 5 hr . starvation in
phosphate buffer , 5 ��dry wt . bacteria/mI . , vi ability 9 4% .
Fig . 1 .
Co cci a r e almost s eparated befo r e invaginations
( 1 ) appear ( Millonig 1 s stain ) .
Figs . 2 , 3 .
Electron dense ribosome pa rticles appear a s
granular dots ( sections were floated o n H 2 0 2 followed by
tr eatment with Reynold ' s stain .
Not e : H 2 0 2 treatment reduces
c ell wall d ensity and increases the contrast of ribosomes ) .
structura l characteri stics of starved organisms
Fig . 4 .
appear simi lar to those in Fig . I ( Mi llonig 1 s stain ) .
Ribo somes are not a s well defined a s in growth pha s e
Fig . 5 .
organi sms ( H 2 0 2 tr eatment , Mi llonig ' s stain ) .
PLAT E 2 .
3
. 5
PLATE 3 .
Organi sms a fter 1 7 hr . starvation in phosphate buffer ,
5 0 �� dry wt . bacteria/mI . , viability 2% .
Fig . 1 .
No ribosome particles are visible ( H O tr eatment ,
Z Z
Mi llonig ' s stain ) .
F igs . Z -4 .
Organi sms showing intrusions o f the membrane in
di r ect contact with the low density nuclea r mat erial ( N ) .
( Reynold ' s stain .
Note : the electron-dense spots are l ead
salt deposits . )
,"
PLATE 3 .
1
2
N
4
Organisms aft er 4 0 hr . starvation in pho sphat e
PLATE 4 .
2+
buffer containing lmM-Mg , 2mg . dry wt . bacteri a/mI . , vi ability
L. l % ( Mi lloni g ' s stain ) .
Fig . 1 .
Bacterial lysi s has occurr ed lea ving the r emains of
Membranes
cytoplasmi c membranes ( eM ) and c el l walls ( CW ) .
t end to r emain intact whi l e cell walls fragment .
Lysed organi sms showing the ext rusion of
F i gs . 1-4 .
cytoplasmi c mat eri al into the ext erna l medium through ruptures
( arrowed ) in the membran e and c ell wall .
PLAT E
4.
••
,
Organisms after 4 8 hr . starvation in phosphate
PLATE 5 .
2+
buffer containing lmM-Mg , lOmM-arginine , casamino a cids ( 1% )
and lOO� � dry wt . bact eri a/mI . , viability 9 2% .
Ribo some parti cles are sti ll present and
Figs . 1 , 2 , 4 .
Lysed
appear to be concentrat ed n ear the cytoplasmi c membrane .
cells are vi sible in Fig . 4 and the lytic pro c ess illustrated
( Sections shown in
in Fig . 3 resembles that shown in Plaw 4 .
all figures were treated with Millonig 1 s stain but for Fig . 2
the section was first floated on H 2 0 ) .
2
PLAT E 5 .
108 .
DI SCUSSI ON
Survi va l of sta rved bact eria
Surviva l mea sur ement s .
The sli de-culture method o f Postgat e
et a l . ( 1 9 61 ) i s well suited for survi val measur ements with
short-chain lactic streptococci such as Streptoco ccus lactis
ML 3 , although vi ability estimates have b e en complicated to some
extent by the chain-forming nature of the o rgani sm and its
t endency to c lump on starvation .
The maximum variance
attributab l e to thes e factors has b een a ssessed and the eff ect
on the trends of survival measur ements could only be small .
The total numbers o f co cci starved in pho sphate buff er remained
constant even when suspensions showed viabi lities hear z ero .
However , aft er prolonged starvation in the presence o f amino
a cids , cell lysis occurred in viable populations as shown by
a fall in the total count and the app earance of lys ed organisms
in el ectron micrographs .
Thi s could have l ed to some high
viability estimates since lysed organi sms may not a lways be
counted on s lide-cultures .
No incr ea s e in c ell mas s or numbers was obs erved in any
starvation conditi ons .
The r eport that S . faecali s di d not
grow when ' starved ' in th e presence of glucos e and casamino
a cids ( Walker & F o r rest , 1 9 64 ) i s surpri sing in vi ew of the
fact that S . lacti s , whi ch has a more r estricted biosynthetic
capacity, gives limited growth under these condition s .
Although
metabolites were rel ea s ed from S . lacti s o rganisms starved in
pho sphate buffer , it s eemed unlikely that these compounds would
support cryptic growth , even at high cell densities partly
becaus e of the organi sm ' s exacting nutritional r equi r ements and
partly through the absen c e of an appropriate amount o f an energy
substrate .
The very limit ed protein synthesi s , demonstrated
from amino acid inco rpo ration stud i es , and the constancy of total
numbers of co cci in suspensions containing exogenous amino a cids ,
109 .
support the impression that cryptic growth did not occur to any
ext ent under th e experimental conditions adopted in thi s study .
Since the present investi gation has been primarily conc erned with
the survi val of the first 9 0% of the population , th ese possible
complications are unlikely to seriously aff ect the int erpr etation
of results .
Str esses whi ch d evelop during the preparation o f washed
suspensions have been minimi z ed by w�shing and r esuspending c el l s
in a defined chemi cal environment at a constant temperature , and
by incubating suspensions near the optimum survi va l pH in phosphate
buff er containing suffici ent EDTA to r emove toxi c metal ions .
2+
Effect of Mg
on survival .
Addition of Mg 2+ ( O . lmM or mo r e )
produced a marked decr ea s e i n the death rate o f starved S . lactis
o r ganisms , a finding whi ch is in agr eement with the observations
o f Po stgate & Hunter ( 1 9 6 2 ) for Aerobacter a erogenes .
Apa rt from
its potenti a l for reducing the toxic effect of certain metal ions
( MacLeo d
1 9 67 ) , Mg
&
2+
Snell , 1 9 5 0 ;
Abelson & Aldous , 1 9 5 0 ; MacL eo d et al . ,
+
, together with Na and Kf , has been established a s an
important stabil i z er of ribo somes in bact eri al cells ( Bowen ,
Dagl ey & Sykes , 1 9 5 9 ; T emp est , Dicks & Hunter , 1 9 6 6 ) . Other
2+
possibl e functions of Mg
in starved bacteria , including membran e
stabi li z ation , have been di scussed by Strange & Hunter ( 1 9 67 ) .
2+
Possible relationships between the presen c e of exogenous Mg ,
ribo some stability and survival will be dis cussed later .
Eff ect of bact erial density .
Harri son ( 1 9 6 0 ) first observed the
effect of bacterial concentration on sur vi val and r eported an
optimum bacteria l density above whi ch the death rate increa sed .
I f the death o f A . a erogenes at high cell concentrations i s due
to anoxia as Harri son has suggested , then the fi nding that
S . lacti s , a fa cultative anaerobe , di d not show an optimum cell
d ensity for survival would be consi stent .
In general , Postgate
& Hunter ( 1 9 6 2 , 1 9 6 3a ) confirmed the findings of Harri son but th ey
di d not o�serve the ' reversed ' population density effect .
The
discrepancy may be explained if anoxia wa s the caus e of the
increased death rate at high concentrations of organisms since
110 .
Ha rrison ( 1 9 6 0 ) did not a erate the wa shed suspensions , in contrast
to the experimental procedur e of Po stgate & Hunter ( 1 9 6 2 ) .
I n the present investigation , organisms in dense suspensions
( equiva lent to 7 . 8 mg . dry wt . bacteri a/mI . ) lost over 50% of
their cell -bound Mg during a 1 2 hr . starvation period in pho sphate
2+ conc entration ( 0 . 7mM ) in the
buff er , producing a protective Mg
ext ernal medium .
With the equi valent of 0 . 7 7 mg . dry wt .
2+
( O . lmM ) was
ba cteria/mI . , a protective concentration of Mg
establi shed in the external medium but death wa s mor e rapi d than
at the higher cell density .
It is po ssible that irreparable
damage was done to the organi sms befor e thi s effective
concentration was reached .
Results suggest that Mg 2+ excretion
by starved organi sms was a maj o r factor in prolonging surviva l
in dense populations and the observation that the a ddition of
0 . lmM-MgS0 produc ed almost i dentical survival times at all
4
population densiti es i s consistent with this vi ew .
I n experiments on glycerol -accelerated death , Postgate &
Hunt er ( 1 9 64 ) r eported that increased bacterial densities
r educed the death rate .
They tested the possibi lity that
2+
excr eted Mg
may have been r esponsible fo r this effect but they
were unable to det ect Mg by chemical methods in concentrates of
cel l -free buffer solutions in whi ch A . a erogenes had died .
However , added Mg 2+ , at concentrations below the limit detectable
with th eir analytical method , protected organisms against glyc erol­
a c c el erated death ( Po stgate & Hunter , 1 9 6 4 ) .
Therefore Mg
excretion , a lthough not detected , may have been r esponsib l e for
at least part of the obs erved bacterial density eff ect .
.
Although excr et ed Mg 2+ concentrations can be related to the
high ba cteri al density eff ect with S . lactis Postgat e & Hunter
U 9 6 3a ) have demonstrated a bact erial density eff ect in the
presence of added Mg 2+ with A . a erogenes . Webb ( 1 9 6 6 ) observed
Mg 2+ liberation by Baci llus megaterium and E . Coli when these
organi sms were starved in a Mg 2+-free salt solution .
Burleigh
& Dawes ( 1 9 6 7 ) fai led to observe a bacterial density effect with
,
starved Sarcina lutea but thei r lowest density ( 1 mg . dry wt . /ml . )
Ill .
may have been too high to obs erve thi s eff ect .
Effect of added substrates .
All of the fermentable carbohydrates
t ested produced acceler ated death of washed organisms i r respective
o f the growth phase or growth-limiting nutri ent .
Carbohydratea c c el erated death was not caused by the lactate produc ed and wa s
2+
markedly r educed on the addition of Mg .
These results contrast
with the obs ervations o f McGrew & Ma llette ( 1 9 6 2 , 19 6 5 ) and
Clifton ( 1 9 6 6 ) , who r eport ed extended survi val of Escherichia coli ,
2+
' without growth ' , in the presence of glucose and Mg .
Po stgate
& Hunter (l9 6 3 c , 1 9 64 ) observed that the addition of c ertain
growth-limiting nutri ents to starving suspensions of c ertain Gram­
negative bacteria increa s ed the death-rat e .
Thi s applied to
nitrogen - , phosphorus - and carbon-limited populations and it wa s
suggested that thi s ' substrate-a ccelerated death ' wa s a fairly
general phenomenon fo r Gram-negative bacteri a .
A lthough carbohydrate energy sources produced accelerated
death , arginine, whi ch S . lactis converts to ornithine with the
production of ATP ( Korzenovsky & Werkman , 1 9 5 3 , 1 9 5 4 ) , produced
2+
Thi s
an increa s e in survi val time in the presence of Mg
indi cated that the natur e of the energy source may be critical
for surviva l .
It i s notewo rthy that in the absence o f added
2+
Mg , a rginine a c c el erated death .
Other a spects of glucose
and a rginine metabo l i sm wi l l be di scussed later .
The extended
survi va l of S . lacti s ML without growth , in the routi� growth
3
medium minus l a cto s e , vitamins and bicarbonate , can be attributed
2+
to its Mg
and amino acid content .
Death rates were reduc ed when
Eff ect of temperature and pH .
the incubation t emperatur e wa s lowered , presumably due �to
This conbra sts
r eductions in the rates of degradative processes .
with th e obs erved increa s ed death rates of A . a erogen es at low
t emperatures ( Po stgate & Hunter , 1 9 6 2 ) but death of this organism
may have been due to a s ensitivity to ' cold shock ' whi ch invo lves
damage to the p ermeabi lity regulating systems ( s ee strange & Dark ,
1962 ;
Strange & Postga t e , 1 9 64 ) .
A lthough it i s beli eved that energy i s r equi red for
11 2 .
intracellular pH control in starved bacteria ( Dawes & Ribbons ,
1 9 6 2 , 1 9 64 ) , thi s ha s not b een experimentally demonstrated .
S . lactis had a sharp pH optimum for survival whi ch i s perhaps
consi stent with the absence of an endogenous en ergy source .
I n thi s connection , the additi on o f arginine wa s shown to produce
a marked d ecrease in death rates at all pH values .
At acid pH
values thi s 'eff ect could have been due to a neutrali zing a ction
of the NH produced from arginine metabolism .
However , arginine
3
a l so pro longed survival at alkaline pH and it s eemed likely that
the en ergy produced from arginine metabo li sm was directly
r esponsible for r educing the lethal effect of adverse pH values .
The growth o f lactic a cid bacteria in mi lk normally c eases
when the lactic a cid fo rm ed produces inhibitory acid conditions .
Although growth c eases , the organi sms r emain viable and continue
to ferment the large excess of lactose present but at a much
slower rate .
It is pos sible that the energy produced by the
continuing f ermentation c ould be an important factor for the
surviva l of these organi sms in sour mi lk .
The very low death rate obs erved in the initi al incubation
period followed by the rapid decline in viabi lity of S . lactis
ML in most resuspended systems , suggests that intrinsi c di fferences
3
in survi va l potential b etween individual bact eri a may be slight .
The duration o f thi s period of almost c omplete viability varied
greatly , dep ending on the r esuspension system and the bi o logical
hi story of the population .
The death rate then began to increas e
a n d generally continued a t a rapi d rate .
Changes in starved organi sms
Polymer degradation .
S . lactis did not accumulate detectable
amounts o f polyglucos e or poly � -hydroxybutyrate in conditions
whi ch would be consid ered as favourable for the synthesi s of
thes e res erve materials .
No r eports have appeared in the
Ii t eratur e indi cati;ng the presence of these or other storage
po lymers in lactic str eptococci .
Forr est & Wa lker ( 1 9 6 5a )
r eported that s . fa ecali s synthesi z ed r es�rve material under
11 3 .
c ertain growth conditions .
However , the nature and metabo lism
of these res erves were not defined .
Only one report ha s b een
publish ed on the metabol� o f S . lacti s starved at growth
t emperatur es and thi s claimed that ( a ) ' the organi sm had a
substantia l endogenous respi ration ' and ( b ) ' endogenous lactate
o r succinat � wa s oxidi z ed after a lag ' ( Spendlove et a l . , 1 9 5 7 ) .
These results have not been confirmed in the present
investigation .
It s eems unlikely that la ctate or succinate
could function as endogenous substrates for an organi sm whi ch
is a homolactic ferment er , poss essing no terminal respiratory
syst em .
Whether b a ct eria po s sess reserves or not � the constitutive
materi al of starved organi sms i s ultimately degraded and death
ensues ( see reviews by Dawes & Ribbons , 1 9 6 4 ; Postgate, 1 9 6 7 ) .
Mo st of the decrea s e in c ell mass of sta rved S . lacti s organi sms
could be a ccount ed fo r by RNA and prot ein degradatio n .
The
rates o f RNA breakdown and death were reduced by the additi on of
2+
Mg ; these observations are in contrast to the findings of
Burl eigh & Dawes ( 19 67 ) with S . lut ea .
However , these autho�s
2+
examin ed the effect o f added Mg
with suspensions o f high
ba ct erial density ( 8 . 8 mg . dry wt . /ml . ) and it is po ssibl e that
2+
wa s li berated by the organi sms as thei r RNA was degraded ,
if Mg
2+
the effect of added Mg
on survival may have been ma sked sinc e
RNA appea red to be to some extent expendable .
The protective
2+
effect of Mg
on Gram-negative organi sms under conditio ns of
stress has been demonstrated with bacterial conc entrations o f
2 0 ;U g . d r y wt . /ml . or l ess ( s ee Postgat e & Hunter , 1 9 6 2 , 1 9 6 4 ;
strange & Postgate , 1 9 6 4 ; Strange & Dark , 1 9 6 5 ) .
These
str esses showed population density eff ects so that the response
2+
o f S . 1ut ea to added Mg
may not be basi cally diff erent from
that defined for Gram-negative o rgani sms .
S . 1acti s ha s both
a high RNA content , typi cal of an organi sm grown at a rapid rat e ,
and a high Mg cont ent , whi ch i s consistent with the probable
interdependenc e o f these constituents ( T empest & Strange , 1 9 6 6 ) .
The molar ratio o f RNA/Mg wa s approximately 5 0 , whi ch is of the
114 .
same o rder a s that observed fo r A . aerogenes ( Tempest & strange
1966 ) .
Conditions whi ch accel erat ed RNA b reakdown - such a s buffer
syst ems whi ch ei ther contained glucose alone or di d not contain
2+
also produced incr eased death rates .
How ever , in the
Mg
2+
presen c e of Mg , arginine only slightly suppressed RNA
degradation although it ext ended surviva l .
Hen c e concommittant
_
Although
RNA degradation and death does not occur in all systems .
considerable RNA may be degraded without aff ecting viabi lity , in
agreement with r esults fo r many other starved ba ct eri a ( see
Burlei gh & Dawes , 1 9 67 ) , it seems likely that a degree of
ribo some stabi lity is important fo r survi val o f S . lacti s .
Conditions whi ch accel erated RNA br eakdown in other bact eria
wer e generally mor e lethal ( e . g . s ee Strange & Shon , 1 9 6 4 ;
Strange & Dark , 1 9 6 5 ) and Postgate ( 19 67 ) has concluded that
RNA degradation is a critical pro c ess in the survival of
A . aerogenes .
However , no absolute correlation between the
I n contra st to these
rates of death and RNA breakdown exi sted .
results, the ' starvation-resi stant ' mutants isolated by Harrison
& Lawrence ( 1 9 6 3 ) degraded RNA more rapidly than the wi ld typ e .
I t has b een suggested that RNA breakdown may continue in
viable bacteria a s long as mechani sms fo r po lymer resynthesis
from precursors r emain inta ct ( Burl eigh & Dawes , 1 9 67 ) .
The
present r esults show that conditions producing maximum rates o f
RNA degradation and death a l s o produce increa s ed cell division
lag times of survi ving o rgani sms .
The se lags are probably
di r ectly influen c ed by the amount of polymer degradati on which
has taken pla c e , particularly that of RNA , sin c e the RNA
content of bacteria increa s es with the growth rate ( T empest &
Strange, 1 9 6 6 ) whi l e the rate of protein synthesi s per ribosome
particle i s constant in growing �acteria ( T empest et a l . , 1 9 67 ) .
Mandel stam ( 19 6 3 ) has suggested that th e restoration o f degraded
protein and RNA takes pla c e gradua lly on the resumpti on of growth .
Most of the RNA in bacteria i s found in the ribosome fraction
and Strange et a l e ( 19 6 3 ) r eported that 7 2% of the total RNA loss
11 5 .
f rom starved A . a erogenes was from ribosomes .
Electron
mi crographs of S . lacti s from a log-phase growth cultur e showed
a dense pattern of ribo somes whi ch app ea red to be depl eted on
_
starvation in phosphat e buffer .
No evi dence was obtained for
the catabolism o f ribo s e or ba ses from degraded RNA , in contrast
to findings with most other starved organi sms ( s ee Dawes & Ribbons ,
1 9 64 ) .
The mechani sm of ribo some di saggr egation and RNA breakdown
in starved E . coli wa s studi ed by Wade ( 1 9 61 ; see also Mandelstam
Most of the depolymerase a ctivity wa s a ssociated with the
ribosomes and was present in an inactive state in the pr esence of
2+
Mg .
Two degradative rout es were proposed dep ending on the
2+
2+
presence o f Mg .
The M rout e ( dep endent on low Mg
concentrations ) appeared to involve a phosphodiesteras e and a
po lynucl eotide pho spho ryla se .
The V rout e involved a
ribonucl ea s e whi ch wa s stimulated by ,removal of Mg 2+ .
It appears
2+
that r emova l o f Mg
initiates ribo some disaggregation which
caus es RNAase a ctivation with rapi d breakdown of RNA to a cid­
so luble products .
Gronlund & Campbell ( 1 9 6 5 ) i denti fied
polyrucl eotide pho sphoryla s e in the ribo some fraction of
P . a eruginosa .
This enzyme was only active at low Mg 2+
concent rations which a llowed the 7 0 s ribosomes to di ssociate .
Mo re r ecently, Ben-Hami da & Schl essinger ( 1 9 6 6 ) have conc luded
that the breakdown of RNA was initiated by the destabili zation
o f polyribosomes .
Mo st o f the p rotein lost from starved organisms appeared
in the ext erna l medium a s biuret-po sitive materi a l a lthough the
n et increa s e in total free amino acids indicated that some
protein had b e en hydro lysed .
Protein hydrolysis occurs in many
starved bacteria with subs equent catabolism of the relea s ed amino
a cids ( s e e Dawes & Ribbons , 1 9 64 ) .
A lthough the overall level .
of free a spartate ( and to a lesser ext ent glutamat e ) was reduced ,
no evidence wa s obtained for appreciabl e catabolism of components
I n contra st , th e free
of the free amino acid pool in S . lactis .
amino aci d poo l , and in particular glutamat e , pro vided the main
11 6 .
eAdogenous substrate for starved S . lut ea ( Dawes & Holms , 1 9 5 8 ) .
I t has been ' suggest ed that pre-exi sting proteases are a ctivated
by starvation conditi ons ( Schl essinger & Ben-Hami da , 1 9 6 6 ;
Wil l ett s , 1 9 6 7 ) and that protea se activity i s determined by the
l evels of pro t ein precursors , perhaps amino-acyl sRNA .
Willetts
( 1 9 67 ) had found no co rrelation between the level of the free
amino a ci d poo l and th e rate o f protein degradation .
The cellular anthrone-reacting material and reducing sugar
of S . l a cti s ( whi ch probably occurs mainly in structural
polyme rs ) together with c ellular DNA , wer e not appreciably
degraded in starvation conditions . These r esults are in agreement
with literatur e reports for other starved o rganisms .
However ,
it has been pointed out by Postgate ( 1 9 6 7 ) and Burl eigh & Dawes
( 1 9 67 ) that chemi cal analyses would not detect structural cha nges
in DNA that could cause loss o f viabi lity .
The loss of RNA and prot ein from starved S . lactis appeared
to involve only hydro lyti c rea ctions with the r el ea s e o f products
into the ext ernal medium .
Viabi lity was not immediately
a ffected and there was no evidence for appreci able energy-yi elding
catabolism of endogenous substrat es .
Thi s wa s consi stent with
the f o rmation of only trace amounts of the normal end products o f
f ermentation , namely lactat e , NH and volati le fatty acids .
As
3
expected , t h e uncoupling agents 2 , 4-dinitrophenol a n d a zide had
no eff ect on survi val .
I n contrast , th e additi on of these
compounds to starved A . a erogenes , whi ch has endogenous energy
sources , substantially increased the death rate ( Postgate & Hunter ,
1964 ) .
Fo rrest & Walker ( 1 9 6 5 a ) found that when S . fa ecalis was
grown with limiting energy sourc e and then starved , the ATP pool
whi ch wa s initially small , declined rapidly .
They concluded
that ' no det ectable endogenous metabolism existed . '
On the other
hand , ' organi sms grown with exc ess energy source exhibited
endogenous metabolism ' and thi s could be co rrelated with a much
higher pool concentration o f ATP .
Polymer synthesi s .
Since mac romolecul e synthesis i s an en ergy
117 .
consuming pro c ess , prot ein synthesis in S . lacti s organi sms
starved in pho sphate buffer wa s not expected as no endogenous
energy source was defined .
In fact , the incorporation of valine­
l4
C into c ell prot ein could only be demonstrated i f an exogenous
en ergy source wa s pres ent .
The extent of the incorporation
appeared to be directly related to the amount of the availabl e ATP .
The acceleration o f valine- 1 4 C uptake by the presence o f an added
energy source, was consistent with mechanisms fo r the a ccumulation
o f amino a cids in other bact eria ( see Ho lden , 1 9 6 2b ) .
The ability of starved organi sms to a c cumulate and
14
incorporate va line- C appeared to be correlated with viability
and the rate o f protein synthesis is probably dir ectly
influenced by the extent of RNA regradation .
It has b een suggested
that vegetative bacteria may be able to und ergo ' adaptation ' in
starvation conditi ons , presumably by turnover o f constituents ,
and that this process may be important for survival ( s ee Duguid
& Wilkinson , 1 9 61 ; Wi l l etts , 1 9 67 ) .
This could explain the
decrea s ed death rate of S . lactis in the presence of amino acids
However , the result s
where some protein synthesis takes pla c e .
from t h e present study show that protein synth esi s does not
influence survi val noti ceably .
In fact , chlorampheni col has
b e en shown to r educ e the death rate in some cases .
As well a s
inhibiting protein synthesi s , chlorampheni col may also inhibit
p rotein degradation ( Willetts , 1 9 67 ) and henc e may exert s ome
sparing action on cellular proteins whi ch are essentia l for
survi va l .
Chloramphenico l , on the other hand , had little or no
eff ect on the survi va l of A . a erogenes ( Po stgate & Hunter , 1 9 6 2 ) .
All reported studies involving starved b a ct eria indi cate that
while pro tein degradation may be balanced by synthesi s during
the initi al starvati on p eriod , dimini shing synthesi s and a net
in�rease in cataboli sm eventually results .
Metabo l i sm of a rginine and gluco se .
Both arginine and glucose
were metabo l i z ed at constant rates by starved organisms and th e
theoretical rate of ATP generation was about 7 . 5 times great er
with lOmM�glucose than wi th lOmM-argini n e , in agr eement with the
118 .
r esults o f Fo rrest ( 1 9 6 5 ) for starved S . faecali s .
Thi s rate o f
glucose metaboli sm i s mor e than 5 0 times the rate calculated for
the supply of maint enance energy in starved E . coli ( Marr et a l . ,
1963 ) .
F rom the data given for the growth o f S . lactis it can
b e calculated that growing organi sms should produc e approximately
1 . 2 ;Umo les lactate/mg . dry wt . bacteria/min . whi l e from
glyco lytic a ctivity measurements it wa s found that non-growing
organisms produc ed O . 2 9 �mol es lactat e/mg . dry wt . bacteria/min .
The simplest explanation for the obs erved linear rates o f glycolysi s
and arginine fermentation in starved S . lactis i s that these
processes are not subj ect to feedback control so that ATP was
generated in amounts whi ch were far in exc ess of requi rements .
This explanation i s in agreement with the results of Forrest &
Walker ( 1 9 6 5b ) .
Forr est ( 1 9 6 5 ) found that the constant input o f
ATP t o the p o o l of starved S . fa eca lis during glycolysi s was
eventually balanced by an exponential decay pro c es s so that the
pool l evel ro s e to an upper limit .
When the exogenous glucos e
was complet ely ferment ed the decay process alone operated and
the pool level fell ba"c k to the much lower endogenous base
l evel .
I n a number of starvation envi ronments , the glyco lytic
a ctivity o f S . lactis organi sms fell steadily from the onset
Other
of starvation and wa s not correlated with viabi lity .
workers have , however , found a correlation between the a ctivity
o f the c atabol i c enzymes and survi val in support of an early
hypothesis a ssociating bacterial death with enzyme inactivation
( Rahn & Schro eder , 1 9 41 ) .
For exampl e , Postgat e & Hunter
( 1 9 6 2 ) found that the glyc erol dehydrogena se and glycerol
o xi da s e a ctiviti es of starved A . a erogenes declined in parallel
with viabi lity , whil e the endogenous r espiration rate of the
glycerol -limited organisms wa s not di rectly related and , in fact ,
dec lined more rapidly .
Simi larly, Burl eigh & Dawes ( 1 9 6 7 )
r eported a correlation between the survi va l of a erobically
starved S . lut ea organisms and their ability to oxidi z e
exogenous glutamat e and glucose .
In the present study the
119 .
glycolytic a ctivity of S . lactis was not maintained with
exogenous energy sources or amino acids including arginin e , in
contrast to th e findings o f Walker & Forrest ( 1 9 64 ) and Forrest
& Walker ( 1 9 6 5a ) .
Indeed , added gluco se produced a mor e rapid
decline in glyco lytic a ctivity .
It seemed possibl e that the extended survi val of starved
S . lactis o rgani sms with added a rgini n e , compared with glucos e ,
was due t o the lower rate o f ATP production .
Thi s explanation
may be consi stent with the results of McGrew & Mal l ette ( 1 9 6 2 )
who claimed to have ext ended the survival o f starved E . coli by
the r egula r addition of very sma l l amounts of glucose , although
their experiment a l conditions did not preclude r egrowth .
In
contra st , most other workers have us ed r elati vely high carbo ­
hydrat e/c el l mass ratio s and have not observed extended
survi va l .
The metabolism o f exogenous glucose by S . lactis
produced long division lags and mor e rapid RNA breakdown in
agreement with the r esults of Postgate & Hunter ( 19 6 4 ) and
strange & Dark ( 1 9 6 5 ) with A . a erogen es .
The l atter autho rs
2+
suggest ed that Mg
abolished substrat e-accel erated death by
preventing the a ccumulation of toxic products but such products
have not been defined .
The end products of carbohydrate. and
arginine metaboli sm are not toxi c to S . lactis but unfavourable
imbalances may have been created in the a s so ci ated metabolic
poo ls ( see Introduction ) .
However , the facto rs responsible
for substrate-accel erated death have not been r eso lved .
strange ' s results ( cited in di scussion , Strange & Hunter , 1 9 6 7 )
indi cated that ATP accumulation during glucose metabolism was
suppressed with added Mg 2+ .
In thi s connection , Po stgate &
Hunter ( 1 9 6 4 ) have shown that glycerol-accelerated death could
be l argely abolish ed by uncoupling agent s .
The death rate o f S . lactis when harvest ed from the lactose­
limited growth medium and starved in phosphate buffer , was
accelerated to simil a r degr ees by the addition of either gluco s e ,
galacto s e , fructo s e or lacto s e .
A l l these sugars were probably
metaboli fed via the glyco lytic pathway .
In contrast , the death
120 .
rate of glycerol-limited A . a erogenes was not acc elerated by
added glucose or ribos e ( Po stgate & Hunt er , 1 9 6 4 ) .
Although
these sub strates were shown to be metabol i z ed , there wa s no
indi cation tha t the rate of thei r metabolism was simi lar to
that in organi sms grown with gluco se o r ribos e limitations
where these sub strates a c c elerated the death rate of starved
o rgani sms .
Since the a ct i vity of constituti ve enzymes may
vary a ccording to growth conditions ( Parde e , 1 9 61 ) , it seems
possible that substrates not included in the growth medium may
be metabol i z ed at slower rates 9Y starved organi sms .
However ,
the strain o f A . a erogenes us ed by Strange & Dark ( 19 6 5 ) showed
carbon-accel erated death with carbon sources besid es the one
which had b een used a s the growth-limiting substrate . Glycerol­
and succinat e-accel erated death decrea s ed on r educing the
substrate con c entration and was undet ectable at low
concentrations ( Po stga t e & Hunter , 1 9 6 4 ) .
It could be informative
to r e-examine the eff ect of low glyc ero l conc entrations on
2+
survival in the pr esence of added Mg .
Strange & Dark ( 1 9 6 5 )
have obs erved that increased rates of glyc erol o xi dation
produced increa s ed death rates and hen c e th ere is now a
considerable amount o f evi d en c e whi ch suggests that the rate
of substrate metaboli sm , and the rate of ATP pro duction , may be
critic a l fo r surviva l .
Slow substrat e metabolism may supply
the maintenan c e en ergy r equi r em ent without delet erious effect s .
The metabol i sm o f exogenou s substrates do es not a lways
a ppear to be coupled to the energy r equirements o f starved
o rgani sms and it s eems po ssibl e that the rapid depletion o f
reserve p olymers in some bacteria may present a simi lar
s ituation with r espect t o endogenous sub strates . Burleigh &
Dawes ( 1 9 67 ) have suggested tha t the rapid metabo lism of
polyglucos e may cause sub strate-accelerat ed death .
The fact
that gluc o s e was not limiting i n their growth cultures may not
be inconsist ent with thi s int erp r etation , since substrates apart
from thos e limiting growth may a c c elerate death as discussed
previously .
Reserve polymers may exert a sparing action on
1 21 .
essenti a l c el l constituents but they seem to enhance survi val
only if th ey a r e broken down at comparatively slow rates
( e . g . s ee Strange et a l . , 1 9 61 ; Si erra & Gibbons , 1 9 6 2 ;
Sobek et a l . , 1 9 6 6 ; Z evenhui z en , 1 9 6 6 ) .
However , Strange &
Hunter ( 1 9 67 ) also pointed out that the enhanced survival of
nitrogen-limited A . a ero genes may be due to the relatively
2+
high Mg
cont ent rather than the glyco gen r eserves .
Changes in permeabi lity and ultra structur e .
Polar lipid and
in particular phospholipi d , constitut es the ma in lipid fraction
in most l a cti c acid bact eria ( see revi ew by Kat es , 1 9 6 4 ) .
In
S . faecali s , 9 4% o f th e total lipid was found i n the membrane
fraction ( Vo rbeck & Marinetti , 1 9 6 5a ) . A substantial amount of
pho spho lipid breakdown occurr ed on prolonged starvation of
S . lactis ML .
Since the s e compounds a re known to have
3
important structura l and physio logical roles in bacterial
membranes ( se e r evi ews by Brown , 1 9 64 ; Salton , 1 9 67 ) , any
breakdown of phospholipids may b e expected to impair p ermeabi lity
barri er s .
There was a stea dy l eakage of the intrac ellular amino acid
pool from star ved S . la cti s into the ext erna l medium .
Release
1
4
o f accumulated valine- C from the T CA -solub l e pool of S . lacti s
wa s suppres sed by the presence o f an exogenous en ergy source .
Thi s r esult i s s imilar to the obs ervation made by Gal e ( 1 9 5 3 )
with S . aureus .
I n contrast t o the present r esult s Holden
( 1 9 6 2b ) has suggested that l eakage of th e free amino acid pool
is a common property of starved Gram-negative bacteria in
contra st to th e r et enti on of the pool by Gram-po sitive
o rgani sms .
However , the important factor, may be the presence
of an ener gy supply sin c e organisms with endogenous en ergy
sources appear to r etain thei r free amino acid pools when
starved ( Dawes & Holms , 1 9 5 8 ; Postgate & Hunter , 1 9 6 2 ; Dawes &
Ribbon s , 1 9 6 5 ) .
Conditions promoting r elea s e o f the
intermedi ate metabolic pools may initi ate po lymer breakdown and
increa s e the dea"t h rate and thi s would be consist ent .with the
observation that spermine enhanc ed survival and suppr essed the
122 .
r elea s e of u . v . -absorbing mat erial from starved S . lacti s .
2
As wel l a s promoting ribosome stabi lity , added Mg + may
have an important function in maintaining p ermeabi lity barri er s .
The finding that high EDTA concentrations were lethal may support
this vi ew sinc e EDTA appea r s to r emove c ell wa ll and membrane
Mg 2+ by chelation .
Aft er approximately 10 hr. starvation of
S . lactis in pho sphate buffer containing Mg 2+ , the death rate
began to increase and wa s a ccompani ed by the r el ease o f lacti c
dehydrogen a s e and DNA .
No c ell lysi s was evident from el ectron
mi crographs prepared aft er 17 hr . sta rvation but most of the
The relea s e of
c ells were lys ed a fter 40 hr . sta rvation .
intra c ellular ma cromo l ecul es , b efore lysis was detectable by
el ectron micro scopy , appeared to r esult from a partial rupture
of the cell wa ll and membrane .
The se structur es were
maintained intact for a longer p eriod in the presence of exogenous
amino a cids and lysi s o ccurred as the viabi lity f ell .
In thi s
environment , death may b e a di r ect result o f c ell wa ll o r membrane
degra dation .
Conclusio$.
The exi st ence of minimum growth rates fo r bacteri a
( T empest et al . , 1 9 67 ) implies a state o f minimum subsistence
whi ch must be maintained for survival .
In thi s stat e , essential
polymers are present at the lowest level capable o f initi ating
r egrowth and any b reakdown of th ese components r esults in loss
o f viability .
I t i s now well establi shed that bact eria produc ed
at maximum growth rates may contain mat eria l in exc ess of these
minimum levels so th at considerable degradation may take place
befor e death .
Organisms capable o f only low rates o f growth
and catabolism may have an advantage for survival in starvation
environments .
The products of polymer hydrolysis in most
bact eria a r e metaboli zed producing energy but with S . lacti s
thes e products generally appear to b e r eleased into the external
medium in an und egraded form .
Although it has been suggest ed that maintenance o f ba cteria
in a viable state r equi r es energy , the amount o f energy i s ill­
defined . , S . lactis maintained complete · viability for 1 5 - 2 0 hr .
123 .
2+
when sta rved in buffer containing Mg , yet no absolut e
requi r ement fo r an energy source could be determined .
At the
onset of starvation an AT P pool was probably present and small
amounts of energy may have been produced by endogenous
metabolism especially if S . lactis contains constituti ve
kina ses �or nuc l eotide metabolism ( see Gronlund & Campbell , 1 9 6 5 ).
However , l ea ka ge of the intrac ellular free amino acid pool
o ccurred a s soon as th e o rganisms were starved , there wa s no
appreci able protei n synthesis and , in addition , starved organi sms
were extr emely pH sensitive .
Thi s suggest ed that an appr eciable
endogenous energy sourc e was not involved in maintaining the
viability of o rgani sms starved in phosphate buffer .
It would
appear that these organi sms remained viable as long as the
degradation of po lymers , particularly RNA , had not pro c eeded
beyond some i rr eversibl e point and the present evidenc e suggests
that the death rate was dependent on the pr esenc e o f compounds
2+
promoting po lymer stability .
When Mg
wa s add ed to buffered
suspensions , a suitable exogenous energy source further enhanc ed
survival .
Thi s energy source appeared to suppress the r elease
of the free amino acid pool and a llow limited prot'e in synthesi s
a nd pH control .
I n thi s envi ronment surviva l may then be a
function of cell wa ll and membrane stabi lity .
S . lacti s do es not a ppear to have any surviva l mechani sm
a s a r esult of an endogenous metaboli sm .
The abi lity to
withstand low pH values may be the most important factor for the
survi val o f thi s organi sm in mi lk .
The present r esults provide
information whi ch may b e rel evant to a pplied studi es involving
' chees e sta rter ' propagation and storage where it is important
to produc e organi sms with minimum c ell divi sion lag times and
maximum viabi liti es and rates of lactate production .
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