Download interaction of salinity and temperature on net protein synthesis and

INTERACTION
OF SALINITY AND TEMPERATURE
ON
NET PROTEIN SYNTHESIS AND VIABILITY
OF
VIBRIO MARINUS1~2
Mary F. Cooper and Richard Y. MO&a
Departments
of Microbiology
and Oceanography,
Oregon
State University,
Corvallis
97331
ABSTRACT
The relationship
of temperature
and salinity to protein synthesis was determined for cells
of Vibrio marinus. The critical temperature
of the lesion in protein synthesis increased with
increasing salinity of the growth medium.
Protein synthesis was significantly
inhibited
at
22C at a salinity of 25%,, but not at a salinity of 35%, until the cells were incubated for
20 min at 24C. The thermal lesion did not involve precursor accumulation
mechanisms
rather than protein synthesis at salinities between 25 and 35%0. At 40%0, the uptake of
extracellular
proline by whole cells was inhibited
at 24C and preceded the inhibition
of
precursor into protein.
Total RNA synthesis continued for 50 min at 22C in growth media at salinities between
15 and 35% but at 40% decreased after 20 min of cell incubation.
At salinities between
15%, and 30%,, total RNA synthesis continued at 15 and 22C, but cellular protein synthesis
was inhibited by either temperature
or salinity effects.
Loss of cell viability
at 22 and 25C at salinities of 25 and 35%0 showed that the onset
of cell death occurs simultaneously
with thermal inhibition
of protein synthesis.
INTRODUCTION
The oceanic variables of temperature
and salinity can vary greatly in nearshore
environments where the effluence of freshwater lowers the salinity of the seawater.
Thermal pollution of freshwaters may result in a large increase in the temperature
of the nearshore water, and differences between fresh and seawaters may be even
greater if cold nearshore waters have upwelled. Such changes in the temperature
and salinity of seawater affect the growth,
metabolism, and survival of marine bacteria as well as the rate at which mineralization processes by bacteria can occur. This
paper attempts to deal with the interaction
of salinity and temperature in terms of
one basic reason for their influence on a
marine bacterium.
Stanley and Morita (1968) noted that
the maximum growth temperature of scv1 Published as Technical
Paper No. 3055, Oregon Agricultural
Experiment
Station.
2 This paper was taken in part from a thesis
submitted
in partial
fulfillment
of the requirement for the M.S. degree at Oregon State University. This research was supported
by National
Science Foundation
Grants GB 8761 and GA
28521.
era1 marine bacteria was a function of the
salinity of the medium. In Vibrio murinus
MP-1 the maximum growth temperature
was 21.2C at a salinity of 35%0 and 10X
at 7%0,about 11” difference. To help clarify such variations in the maximum growth
temperature of marine bacteria at different salinities, this investigation was initiated to determine the organism’s ability to
synthesize protein, an essential metabolic
function for its growth.
MATERIALS
AND
METHODS
Media
The SDB agar medium was composed
of polypcptone (BBL), 5.0 g; yeast extract
( Difco ), 3.0 g; Rila marine mix ( Rila
Products, Teancck, N. J. ), 5.0 g; sodium
chloride, 15.0 g; glucose, 0.5 g; succinic
acid, 0.2 g; ferrous sulfate, 0.01 g; Bactoagar (Difco),
15.0 g; distilled water, 1
liter. The pH was adjusted to 7.5 with
NaOH.
Succinate salts medium ( SSPU ) consisted of succinic acid, 2.5 g; ammonium
sulfate, 2.0 g; K2HP04, 4.0 g; KH2P04,
0.5 g; MgSO *7H20, 0.1 g; vitamin solution, 1 ml; trace elements, 1.0 ml; proline,
SALINITY-TEMPERATE
EFFECTS
10 mg; uracil, 10 mg; NaCl was added in
the following concentrations for each salinity:
15%0, 8.5 g; 20%0, 13.7 g; 25& 18.8
g; 30%0, 24.0 g; 35%0, 29.2 g; 40%0, 34.5 g.
The total volume of each medium was adjusted to 1,000 ml with distilled water.
The pH was adjusted to 7.8 with NaOH.
The vitamin solution contained nicotinamide, 400 mg; thiamine, 100 mg; pyridoxinc HCl, 100 mg; riboflavinc,
25 mg;
biotin, 1 mg; calcium-d-pantothenate,
100
mg; and distilled water to a volume of
1,000 ml. The trace element solution contained the following rcagcnt-grade chemicals: CaC12.2 H20, 200 mg; FeC& *6H2O,
100 mg; KBr, 100 mg; KI, 10 mg; MnC12.
d&to,
100 mg; SrC$ *6H2O, 100 mg;
HZBOS, 100 mg; CoC12*6H20, 100 mg; and
distilled water to a volume of 100 ml.
The pH of all media was 7.4 after sterilization by autoclaving at 2 atm for 15
min.
Organism
The obligate marine psychrophile,
Vibrio marinus MP-1, was isolated by Morita
and Haight ( 1964) from the North Pacific
at a water temperature of 3.24C and classified by Colwell
and Morita
( 1964).
Monthly
transfers of the stock culture
were inoculated onto SDB agar slants and
incubated at 5C. New working stock cultures were established every 2 weeks by
loop transfer from the SDB agar stock culture into 50 ml of SSPU ( 35%0) medium in
a 250-ml crlenmeyer flask, Incubation was
at 5C in a psychrotherm incubator (New
Brunswick Sci. Co.) with the reciprocal
shaker set at 100 strokes/min,
This working stock culture was maintained by a 10%
inoculum (v/v)
into fresh SSPU (35so)
medium cvcry 3 days.
Growth
of the culture
Five milliliters of the working stock culture were inoculated into 50 ml of SSPU
medium in a 250-ml erlcnmeyer flask and
incubated in the psychrotherm for 30 hr at
15C.. Ten milliliters
of this culture were
inoculated into 100 ml of fresh medium in
ON
PROTEIN
SYNTHESIS
557
a 5OO-ml sidearm flask and incubated
the psychrotherm.
Preparation
in
of log phase cell suspension
Fifty milliliters of a 12-hr culture of log
phase cells in SSPU medium were filtered
in the cold at 4-7C onto a sterile Millipore
filter (47 mm, 0.45 JU) previously rinsed
with sterile SSPU medium. Filtration was
facilitated
by vacuum with the negative
prcssurc not exceeding 25.4 cm of Hg.
The filtered cells were washed with 5 ml
of SSPU medium at 4-7C and resuspended
in fresh SSPU to an OD of 0.125 estimated
at 425 rnp on a spectrophotometer
(Beckman DB ) in a quartz cuvette with a path
length of 1 cm against a distilled water
blank. For ccl1 preparations at salinities
of 25-40so, 12-hr log phase cultures were
filtered, rinsed, and resuspended in SSPU
medium at the same salinity. To obtain
sufficient cells for studies run at 15 and
20%0, cultures were grown in SSPU medium at a salinity of 25%0. The 12-hr cultures were washed and resuspcndcd with
SSPU medium at the lower salinities. This
latter procedure should not interfere with
our experiments since MacLeod
(1968)
has presented data that the intracellular
Na+ and Cl- are the same as the extracellular Na+ and Cl- except when the suspending menstruum
is at a very low
salinity.
Protein synthesis studies
L-Proline UL-C-14 (Int. Chem. and Nuclear Corp.) was added to a cell suspension in log phase at l5C in a I25-ml erlenmeyer flask to an activity of 0.05 &i/ml.
After warming the ccl1 suspension to each
experimental temperature, 4-8-ml portions
were pipetted into test tubes that were
temperature equilibrated
in the polythermostat. Incorporation
of labeled proline
into protein was determined by a modification of the method described by Kennel1
( 1967). At 10-15-min intervals 0.5 or 1.0
ml of cell suspension was pipetted into an
equal volume of trichloroacetic
acid (20%
w/v) in a 1.6 X 15 mm test tube placed in
crushed ice. The sample size was constant
558
MARY
F. COOPER AND RICHARD
throughout each cxpcriment. The contents
of the tube were mixed thoroughly,
and
cold trichloroacctic
acid (5%) was added
to bring the total volume to 3 ml. This
mixture was kept on ice for 30 min, Each
tube was covered with a marble and
placed in a water bath at 82C for exactly
30 min. The tubes were cooled and the
hot acid-insoluble material was recovered
by filtering the mixture through a 22-mm
microfiber-glass disk prcfiltcr (AP20, Millipore) previously soaked with trichloroacetic acid ( 10%)) and the rinse material
was filtered through the glass prcfilter.
The prefilter was rinsed once with 4 ml
of cold trichloroacetic acid (10%) and twice
with 5 ml of cold ethanol (70% v/v). The
filters wcrc placed in counting vials, dried
under a heat-lamp for 2 hr, and covered
with 5 ml of scintillation
fluor solution
composed of 2,5diphenyloxazolc
( PPO ) ,
4.0 g; dimethyl
1,4-bis-2-( 5-phcnyloxazolyl ) -benzene ( POPOP) , 0.30 g; toluene,
1,000 ml. Radioactivity was measured in a
liquid scintillation spectromctcr ( Tri-Carb
model 300, Packard Instr. Co.), A 14Cprolinc sample was used to dcterminc the
optimal instrument settings. A glass prefilter rinsed as a sample was used to
determine background.
Proline was used in these studies bccause, in our initial attempts to formulate
a defined medium, WC found proline was
one of the amino acids rcquircd.
Uptake studies
The uptake of 14C-proline by whole cells
used in the protein synthesis studies was
determined by filtering 0.5 ml of ccl1 SUSpension on a 25mm cellulose membrane
filter (0.45 p, Millipore)
presoaked with
cold 75% seawater containing 26.3 g Rila
marine mix in 1,000 ml of distilled water.
The filtered cells were immediately rinsed
with 2.0 ml of cold 75% seawater. The
filters were placed in counting vials, dried,
and covcrcd with 5 ml of scintillation
fluor solution, Uptake of 14C-proline was
estimated by counting radioactivity
as dcscribed above.
Y. MORITA
1.6-
L./ A ,
0
6
12
TIME
I6
24
,
30
(HOURS)
FIG. 1. Growth c~~rvcs of Vibrio marinus Ml?-1
in SSPU medium at 25 and 35%. Volume of cell
suspension was 1.0 ml.
Studies of ribonucleic
acid synthesis
Uracil UL-H-3 (5.61 Ci/mM; New England Nuclear Corp.) was added to a 15C
suspension of log phase cells to an activity
of 1 &i/ml.
Samples were treated as dcscribed above for protein synthesis except
that trichloroacetic acid (10%) was used instead of trichloroacctic
acid (5%). The 30min incubation at 82C, used to rcsolubilizc
RNA, was eliminated.
The appropriate
settings for the scintillation
spcctromcter
were determined with a sample prcparcd
for RNA analysis.
RESULTS AND DISCUSSION
In preliminary
investigations we cstablished constant experimental conditions to
determine the effects of the variable parametcrs of tcmpcraturc and salinity. The
only constituent that varied in concentration in the synthetic SSPU medium at the
six salinities was NaCl. A medium of dcfined composition allowed a more precise
calculation of the oceanographic paramcter of total salinity than a complex mc-
SALINITY-TEMPERATURE
EFFECTS
16
I
0
1
30
TIME
60
90
(MIN)
FIG. 2.
Incorporation
of W-proline
( x 10”
count min-l)
into protein
during incubation
of
Vibrio marinus MP-1 in SSPU medium at 25%,.
Volume of cell suspension was 1.0 ml.
dim11 containing large quantities of organic materials. The use of succinic acid
rather than glucose eliminated the ncccssity for separate autoclaving and subscquent mixing of carbon source and salts
of the medium. Unlabeled L-prolinc and
uracil were added to the SSPU medium
to ensure the activation of cellular accumulation systems, if present in V. mnrinus
MP-1, before the introduction
of radiotracer compounds. This shortened an initial time lag in the uptake and incorporation of labeled precursors into whole cells
and cellular macromolecules.
All cell suspensions studied at the six salinities were taken from the middle of log
phase growth, Growth curves in the mcdium at salinities of 25 and 35%0 were similar, with the cultures entering log phase
growth about 4 hr after inoculation and
with inflection points, indicating the end
of exponential growth, at 21 hr ( Fig, 1).
The size and morphology of cells grown
at different salinities can vary considcrably and thereby result in vastly different
ccl1 numbers corresponding to the same
ON PROTEIN
SYNTHESIS
559
optical density f ccl1 resuspcnsions (Gibbons and Payn 1961). To obtain a constant number of” resuspended cells for each
salinity investi latcd, it was important to
establish the rc ationship bctwecn optical
density and to1 al cell count of resuspcnsions made fro
cells grown at different
I&I
salinities. Studies of OD vs. cell numbers
showed that counts of cell resuspcnsions
in SSPU medium at salinities bctwccn 25
and 40so resulted in correspondingly identical values of OD of cell rcsuspcnsions.
Incorporation
of prolinc into protein during incubation
of V. mnrinus MP-1 in
SSPU (25%o) medium continued for 90
min at 15 and 20C but was negligible at
25C (Fig. 2). Simi 1,qr results were obtained
from comparative protein synthesis studies
at salinities of 30, 35, and 40so. At all four
salinities, the rate of protein synthesis at
20C was about equal to or greater than
the rate at 15C. These studies established
that the critical temperature range of the
thermal lesion in protein synthesis is bctwecn 20 and 25C for salinities of 2540%,. Since growth at 15C was extremely
poor in SSPU (207&) medium, comparative
protein synthesis was not studied at this
salinity.
Subsequent studies of protein synthesis
at 1” intervals in the 5C critical range arc
reported for salinities of 25, 30, 35, and
40%0 ( Fig. 3). Al so included arc the rcsuits oE concurrent precursor uptake studies. At salinities of 30 through 35so, the
uptake of precursor into whole cells continued after protein synthesis had dccrcascd. At 40%0, the dccrcase in uptake
of precursor at 24 and 25C is significant
as compared to uptake at 20C and prccedes by 25 min the dccrcasc in incorporation of label into protein, A considerable
dccrcasc in protein synthesis, and in prccursor uptake, occurs at 22C in cells suspended in SSPU (257&o) medium (Fig. 3).
In SSPU (307&) medium, the thermal inhibition of protein synthesis occurs in a
gradual gradient of decreasing rates betwcen 20 and 24C, with protein synthesis
still apparent after 40 min at 24C. In
SSPU 35%0, protein synthesis dots not dc-
MARY
560
F. COOPER AND RICHARD
25 %0
0
Y. MORITA
30 %0
I6
n
PROTEIN
PROTEIN
8
I2
6
8
4
I4 -
3 .
UPTAKE
UPTAKE
0 2oc
n
0
IO
9
m 22c
0
6
+
n
;#
0
y51ME
30
(MIN)
3 5700
IO-
1
45
20-23C
8
5O
TIME
8-
40
30
20
IO
(MIN)
20-
4 0 %0
23C
PROTEIN
PROTEIN
7-
6-
5.
7-
I
UPTAKE
5L
0
TIME
(MIN)
L
IO
30
20
TIME
I
40
(MIN)
FIG. 3. Incorporation
into protein (top of each graph) and whole cell uptake (bottom of each graph)
of “C-proline
( x lOa count min-l) by Vibrio marinus MP-1 incubated
in SSPU medium at various
salinities.
Volume of cell suspension was 1.0 ml.
SALINITY-TEMPERATURE
EFFECTS
crease until after 20 min of incubation at
24C. These studies may indicate that at
different salinities of growth medium, differcnt mechanisms are involved in the inhibition
of precursor incorporation
into
protein at 24C. Since the decrease in uptake precedes the decrease in incorporation of precursor into protein at 40%0, the
apparent decrease in protein synthesis at
24C may result from inhibition of cellular
transport mechanisms. A salinity of 4O$L
appears to prevent the uptake of extraccllular amino acids at temperatures that did
not prevent uptake at the lower salinity of
35%0. The lesion at 24C in SSPU ( 40%0)
medium may not directly
involve the
protein-synthesizing
system. At salinities
of 25-35& precursor uptake continues after thermal inhibition of protein synthesis,
thus suggesting a lesion in the cellular
protein-synthesizing
system. The thermal
lesion in protein synthesis occurs at lower
temperatures in lower salinities of growth
medium. Thus a temperature-salinity
relationship is involved in the thermal inhibition of protein synthesis.
The site of the thermal lesion in cellular protein synthesis may be either in
transcription
of m-RNA, involving
ccllular control mechanisms, or in translation
to protein involving heat sensitive synthetase enzymes, ribosomes, or transferase
enzymes. To determine which of these cellular events is initially inhibited by heat,
we studiedthe synthesis of RNA and protein concurrently at 15 and 22C in SSPU
medium at salinities from 15 through 40s0
( Fig. 4). At salinities of 20 through 35%0,
total RNA synthesis was greater at 22C
than at 15. Continued incorporation
of
uracil into RNA at 15 and 22C may indicate that no lesion occurs in uptake of this
RNA precursor into whole cells, In SSPU
( 40%,) medium ( Fig. 4)) incorporation of
uracil into RNA decreased after 10 min of
incubation at 22C compared to RNA synthesis in the control at 15C. This decrease
may reflect either an inhibiton
of RNA
synthesis at 22C or an inhibition of uracil
uptake similar to that observed for prolinc
at 24C and 407L. At 22C, the decreased
ON PROTEIN
SYNTHESIS
561
rate of protein synthesis in SSPU medium
at 25 and 30%0 and the continuation
of
protein synthesis at 35 and 40%0 duplicate
the results of the critical temperature studits. In SSPU ( 15%0) medium, which dots
not support growth, protein synthesis is
negligible at 22C, but continues for 30 min
at 15C and then decreases in rate. Similar
results are seen in SSPU (20%0), but slight
protein synthesis occurs for 30 min at 22C.
The decrease in the rate of protein synthesis at 15C after 30 min of incubation in
SSPU medium at 15 and 20%0 may either
result in, or result from, the inability
of
cells to grow at these low salinities. Thus,
at growth medium salinities of E-30%0,
although total RNA synthesis continues at
both 15 and 22C, cellular protein synthesis
is inhibited by either temperature or salinity effects. These results may explain the
low maximum temperature for growth of
10.5C demonstrated by Stanley and Morita
(1968) in V. murinus MP-1 cells in mcdium at a salinity of lo%,.
The correlation between the time of inhibition of protein synthesis and the onset
of cell death was determined in SSPU at
salinities of 25 ( Fig. 5) and35%0 ( Fig. 6).
The onset of cell death and the inhibition
of protein synthesis both occur after about
40 min of incubation
at 22C in SSPU
(25%,) medium (Fig. 5). At 25C in the
same medium, both cell death and the
inhibition
of protein synthesis begins immediately.
In SSPU ( 35%0) medium, the
viability loss is very low at 22C until after
2 hr of incubation at which time the cells
undergo a rapid loss of viability
( Fig, 6) ..
Concurrent protein synthesis at 22C continues for 2 hr at a lower rate than in the
control at 15C and then completely stops
at the same time the rapid loss of viability
occurs. The loss of cell viability is immcdiate at 25C in SSPU ( 357L) medium and
dccrcases to 25% of the original viability
after 2 hr of incubation.
Protein synthesis
occurs at a low rate at 25C for 1 hr and
then stops completely. These data directly
correlate the loss of ccl1 viability with the,
thermal inhibition
of protein synthesis at
25 and 35%0. Controls run at 15C show
562
MARY
F. COOPER AND RICHARD
Y. MORITA
3
7
PROTEIN
l5C
PROTEIN
x
/
:
;6
c:
-A-A0
IO
.
20
TIME
TIME
30
(MIN)
(MIN)
40
50
0
1
20
IO
TIME
TIME
.
30
(MIN)
(MIN)
.
40
8
50
-rt
H
0
.
IO
.
20
TIME
TIME
.
30
.
40
,
50
(MIN)
(MIN)
FIG. 4. Incorporation
of ‘H-uracil
(X lo2 count min-l) into RNA (top of each graph) and 14C-proline
( x lo2 count min-l) into protein (bottom of each graph) during incubation
of Vihrio marinus MP-1
in SSPU medium at various salinities.
Volume of cell suspension was OS ml.
continued cell viability
and protein synthesis for the entire experimental period.
Data from Morita and Albright (1968)
also demonstrated that a temperature 1 or
2C above the maximum for growth of V.
marinus MP-1 does not stop protein synthesis. The experiments of Malcolm (1968,
1969) were performed 5C above the maximum tcmperaturc for growth of his organism; we beIievc that this extreme tcmper-
aturc may mask more subtle immediate
events that Iead to thcrma1 death of a ceI1.
The data indicate that the thermal lcsion in protein synthesis is at the transcription level. Farrell and Rose ( 1965,
1967), Langridgc and Morita ( 1966)) and
Mathemeier ( 1966), dcscribcd enzymes as
the most heat-labile cell constituents.
In
an obligate psychrophile, Malcolm ( 1968,
1969) demonstrated the tempcraturc scn-
SALINITY-TEZMPERATURE
16-
25%0
EFFECTS
+I5 c
ON PROTEIN
16[ 35%0
I4 -
I4 PROTEIN
+
SYNTHESIS
I2 -
+
563
SYNTHESIS
PROTEIN
/
+I5
c
+
SYNTHESIS
/
IO +
A-
e-
A-A-42
c
1
/+
y/z
4-
-.-.
-.-.-.25C
1
I
VIABILITY
+I5
c
l
mm-+
160 -
A-A<
VIABILITY
r-A-W.A-A22
,:\
C
0
\
200
1
20
l
I ,25
40
TIME
C
1
60
I
80
1
100
1
120
(MIN)
FIG. 5.
Protein
synthesis (14C-proline
x lo2
count min-l)
and cell viability
during incubation
of Vibrio mariners MP-1 in SSPU medium at 25%.
Volume of cell suspension was 1.0 ml.
sitivity of three aminoacyl-t-RNA
synthctase enzymes (those for glutamic acid,
histidine, and prolinc ) and one t-RNA spctics (that cognate for glutamic acid).
Heated psychrophilc
cnzymcs would not
amino-acylate t-RNA molcculcs from any
organism. However, heated psychrophile
t-RNA was aminoacylatcd by hctcrologous
mcsophile or thermophilc
synthctase cnzymcs but not by its own Lmheated synthctasc enzyme. Malcolm suggested that
the conformation
of t-RNA
molecules
changes with tcmpcrature, and the conformation of recognition sites for psychrophilic synthetasc enzymes may differ from
those for mesophilic or thcrmophilic
enzymcs . Sinclair and Grant (1967) have
reported a similar thermolabile
receptor
activity of the t-RNA cognate for leucine
from an obligatcly
psychrophilic
yeast
exhibiting
a thermal lesion in protein
synthesis.
Biochemical studies of Escherichiu coli
mutants with temperature sensitive phcnylalanyl RNA synthctascs demonstrated that
0
30
60
TIME
SO
120
150
I80
(MIN)
FIG. 6.
Protein synthesis
(14C-proline
x lo2
count min-l)
and cell viability
during incubation
of Vibrio marinus MP-1 in SSPU medium at 35%,.
Volume of cell suspension was 1.0 ml.
the mutant cnzymcs consisted of two, noncatalytic subunits, each half the size of
the wild synthetase (Bock 1968). Bock
(1968) suggested that the dissociation or
weakening of subunit interactions causes
the thermolability
of this enzyme. In view
of the previously discussed examples of
the specific rcquiremcnt
for Na+ (Goldman et al. 1963; MacLcod 1965) and of
the salt activation of cnzymcs from halophilic organisms (Drapeau and MacLeod
1963, 1965; Rhodes and Payne 1966), it
seems possible that thermally
induced
changes in structural and functional areas
of synthctasc enzymes or t-RNA molccules may be protected by the binding of
Na-’ ions to these molcculcs. The rcduction of intramolecular
electrostatic repulsion by salt may prevent the weakening
of thermally incrcascd intramolecular clcctrostatic repulsive interactions and subscqucnt conformational
changes.
Since all RNA synthesis studies in these
564
MARY
F. COOPER AND RICHARD
experiments determined only total RNA
synthesis, it is possible that the lesion is
at the transcriptional level, preventing the
synthesis of m-RNA while allowing the
synthesis of other RNA species to continue. This would result in a subsequent
decrease in the synthesis of cellular protcin. Since turnover of m-RNA occurs in
protein-synthesizing
cells and the amount
of m-RNA present in cells at any time may
be low compared to the amount of total
cellular RNA, differences in the amount of
m-RNA synthesized at 22 and 15C may not
be detected in the experimental system.
A number of cellular control mechanisms may be implicated in the thermal
damage to V. marinus. Several investigations have demonstrated regulatory
systems that are temperature sensitive (Gallant and Stapleton 1963; Horiuchi et al.
1961) . Gallant
and Stapleton
( 1963)
showed that a system controlled at low
temperatures by inorganic phosphate repression undergoes constitutive
alkaline
phosphatase synthesis at high temperatures due to a thermosensitive aporepressor synthesizing system. Morita and Albright (1968) demonstrated that V. marinus MP-1 synthesized more RNA, DNA,
and protein at 21 than at 15C-the
opfor growth of the
timum temperature
organism. More total RNA was also synthesized at 22 than at 15C at salinities of
20 through 35s0. These results are similar
to those reported by Harder and Vcldkamp ( 1967) in their continuous culture
study of an obligate psychrophile. At temperatures above the optimum for growth,
more total RNA and protein were synthcsized to compensate for thermally
impaired protein synthesis. The optimum
temperature for respiratory enzyme activity was above the maximum growth tem-perature of the organism. In V. marinus
cells, the synthesis of more total protein
at 20C than at 15 may result from the
synthesis of more total RNA in response
to both the partial thermal inactivation of
protein-synthesizing
systems and the inactivation of cellular enzymes.
The data presented here definitely
es-
Y. MORITA
tablish a relationship between temperature
and salinity in protein and RNA synthesis
in V. ma&us, which in turn can be extrapolated to the nearshore environment.
REFERENCES
BOCK, A. 1968. Relation bctwecn subunit structure and temperature-sensitivity
of mutant
phenylalanyl
RNA synthetases of Escherichia
coli.
Eur. J. Biochcm. 4: 395-400.
COLWELL, R. R., AND R. Y. MORITA.
1964. Rcisolation
and emendation
of description
of
Vibrio ma&w
(Russell)
Ford. J, Bacterial.
88 : 831-837.
DWEAU,
G. R., AND R. A. MACLEOD.
1963.
Sodium dependent active transport of alpha
aminoisobutyric
acid into cells of a marine
Biochem. Biophys. Res. Compseudomonad.
mun. 12: 111-115.
-,
AND -.
1965. A role for inorganic
ions in the maintenance
of intracellular
solute concentrations
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