Download Autotrophic growth on methanol by bacteria isolated from activated

239
FEMS MicrobiologyLetters 5 (1979) 239-243
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
A U T O T R O P H I C G R O W T H O N M E T H A N O L BY B A C T E R I A I S O L A T E D F R O M A C T I V A T E D
SLUDGE
NINA V. LOGINOVA and Y.A. TROTSENKO
Institute of Biochemistry and Physiology of Microorganisms, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region,
142292. U.S.S.R.
Received 8 December 1978
1. Introduction
Methanol is a simple reduced Ct-substrate that is
utilized by many different microorganisms. The pathway of carbon assimilation from methanol has been
studied extensively, particularly with respect to the
question whether C rcompounds are assimilated as
CO2 or at a more reduced level.
Three cyclic mechanisms for the assimilation of
Cl-compounds are known, namely the serine pathway, the ribulose monophosphate pathway and the
ribulose bisphosphate pathway [1,2]. The first two
routes operate in methylotrophic micro-organisms
which assimilate methanol carbon mainly as formaldehyde. The ribulose bisphosphate cycle is characteristic for photo- and chemolithotrophic bacteria,
which assimilate CO2 and use methanol either as a
reductant or as a source of energy [3-7].
In the serine pathway methanol carbon is incorporated by hydroxymethylation of glycine to form
serine, followed by its conversion through hydroxypyruvate, glycerate, phosphoglycerate to phosphoenolpyruvate. The latter is carboxylated and transformed into malate. The cleavage of malate leads to
net synthesis of acetyl-CoA from two Crunits
(formaldehyde and COz). Hydroxypyruvate reductase, serine-glyoxylate aminotransferase and ATP-,
CoA-dependent malate lyase are considered to be key
enzymes of the serine pathway [1,2].
The key reactions of the ribulose monophosphate
cyclase are hydroxymethylation of ribulose-5-phosphate with the formation of the specific product,
D-arabino-3-hexulose-6-phosphate, which undergoes
isomerization to fructose-6-phosphate. These reactions are catalysed by 3-hexulosephosphate synthase
and phospho-3-hexuloisomerase, respectively [2,8].
The subsequent cleavage of fructose-6-phosphate
leads to synthesis of (phospho)-trioses.
The unique reactions of the ribulose bisphosphate
cycle - phosphorylation of ribulose-5-phosphate and
carboxylation of ribulose-1,5-bisphosphate with formation of phosphoglycerate - are catalysed by phosphoribulokinase and ribulose bisphosphate carboxylase. The levels of the enzymes necessary for operation of the above Cl-assimilation pathways are generally much higher in cells grown on media with
C i-than during growth on Cn-compounds.
So far only three cases of autotrophic carbon
assimilation during growth on methanol by nonphotosynthetic bacteria i.e. Paracoccus denitrificans
[5], Thiobacillus novellus [6], and Microcyclus
aquaticus [7] have been reported. This could be
taken to indicate that the Calvin cycle has a limited
distribution amongst microorganisms growing on
methanol or other reduced Crsubstrates which might
be due to a higher energy expenditure of the Calvin
cycle as compared to the serine pathway and the
ribulose monophosphate cycle.
This paper presents the results of an enzymic
study of primary and intermediary metabolism of
methanol by three bacterial strains of different genera,
which were isolated from activated sludge [9]. The
data indicate that these organisms utilize methanol as
an energy source and fix CO2 via the Calvin cycle.
240
Achromobacter {Bacterium sp. 1L), Pseudomonas sp. 8 and Mycobacterium sp. 50 were kindly
supplied by Professor E.N. Kondratieva (Moscow
State University, Department of Microbiology, Moscow, USSR). The organisms were maintained on
methanol or glucose agar slopes and were inoculated
into the appropriate media. After 3 - 5 successive
transfers the cells were used for enzymic studies. All
liquid cultures (200 ml) were grown in 700 ml erlenmeyer flasks with a basal mineral medium on a
rotary shaker (120 rev./min). The medium contained (g/l): KH2PO4 - 2.0; (NH4)2SO4 - 2.0;
MgSO4 • 7H20 - 0.025; NaC1 - 0.5; FeSO4.7H20 traces, 7.2. The carbon sources added to the medium
were sterilized separately. The final concentrations
were: 0.5% (v/v) for methanol and 0.3% (w/v) for
glucose.
For autotrophic growth the mineral medium and
gas mixture 70% H2 : 10% CO2 : 20% O2 previously
described [7] were used.
NAD or phenazine methosulphate [12], hydroxypyruvate reductase EC 1.1.1.29 NADH- or NADPHdependent, as well as serine glyoxylate aminotransferase [13 ], ATP malate lyase and isocitrate lyase
EC 4.3.3.1 [ 14], 3-hexulose phosphate synthase [ 15 ],
ribulose bisphosphate carboxylase EC 4.1.1.39 [7],
hexokinase EC 2.7.1.2. [16], glucose-6-phosphate
dehydrogenase EC 1.1.1.49 and 6-phosphogluconate
dehydrogenase EC 1.1.1.48 [ 16], fructose diphosphate
aldolase EC 4.1.2.13 [ 17 ], phosphogluconate
dehydrase EC 4.2.1.12 and phospho-2-keto-3-deoxygluconate aldolase EC 4.1.2.14 [ 18 ], pyruvate dehydrogenase EC 1.2.4.1 and a-ketoglutarate dehydrogenase EC 1.2.4.2 [19], citrate synthase EC 4.1.3.7
[20], isocitrate dehydrogenase EC 1.1.1.41 and
EC 1.1.1.42 [21 ], malate dehydrogenase EC 1.1.1.37
[22].
Spectrophotometric assays were performed with a
Specord UV VIS spectrophotometer and radioactivity
was counted in a liquid scintillation spectrometer
SL-30 Intertechnique. Enzyme activities are expressed
as nanomoles of substrate transformed in 1 min per.
mg of protein. Protein was determined by the method
of Lowry [23].
2.2. Preparation o f cell-free extracts
2.4. Chemicals
For the preparation of cell-free extracts organisms
were harvested from the exponential phase, washed
once with 50 mM Tris-HC1 or potassium phosphate
buffer pH 7.5, suspended in appropriate buffer, containing 0.001 M dithioerythritol. Concentrated cell
suspensions were disrupted by passing once through a
Hughes pressure cell, pre-cooled to -30°C, at a
pressure of 3000 kg/cm 2. After slow thawing, cells
and debris were removed by centrifugation at 9000 g
for 20 rain. The supernatant obtained after a second
centrifugation (30000 g, 50 min) was used as the cellfree extract.
Purified biochemicals enzymes and coenzymes
were obtained from Sigma, Serva, Calbiochem, KochLight, Boehringer and Reanal.
2. Materials and Methods
2.1. Organisms and growth conditions
2.3. Enzyme assays
Cell-free extracts were assayed at 30°C for the
following enzyme activities by published methods:
methanol dehydrogenase EC 1.1.99.8 [10], methanol
oxidase [11], formaldehyde dehydrogenase (NAD+/
GSH linked) EC 1.2.1.1 or phenazine methosulphate
linked and formate dehydrogenase EC 1.2.1.2 with
3. Results and Discussion
The three bacteria used in this study were isolated
from activated sludge via enrichment in methanol
media [9]. Optimal growth conditons were: t = 2 8 30°C, pH 7.0 and a methanol concentration of 0.5%
(v/v). Apart from methanol our isolates grow well in
liquid media with a variety of polycarbon compounds as the sole carbon and energy source such
as alcohols, sugars and organic acids. Methane, methylated amines, formaldehyde and formate did not
support growth. It was shown by us that the
organisms required biotin (30/ag/1) during growth on
methanol.
To determine at which oxidation level carbon is
assimilated by Achromobacter sp. 1L, Pseudomonas
241
TABLE 1
Specific activities of enzymes involved in primary metabolism of C1-compoundsin extracts of methanol-and glucose-grownbacteria
Enzymes were assayed by the methods indicated in Materials and Methods.
Enzyme
Hydroxypyruvatereductase
NADH
NADPH
Serine-glyoxylate aminotransferase
NAD(P)H
ATP malate lyase
Hexulose phosphate synthase
Ribulose bisphosphate carboxylase
Formaldehyde dehydrogenase
NADTGSH
PMS
Formate dehydrogenase
NAD÷
PMS
Enzyme activities (nmol min -1 mg protein -1 )
Achromobacter 1L
Pseudomonas 8
Mycobacterium 50
Methanol
Methanol
Glucose
Methanol
Glucose
Glucose
226
320
290
290
115
210
124
200
414
227
462
277
0
0
0
420
0
0
0
0
0
0
0
315
0
0
0
0
0
0
0
271
0
0
0
0
70
164
0
226
82
100
0
140
84
209
0
376
1310
106
120
0
1650
120
159
0
550
83
51
0
sp. 8 and Mycobacterium sp. 50, the activities of key
enzymes involved in primary C 1-assimilation were
assayed in cell-free extracts of the bacteria grown on
methanol or glucose. The results are shown in Table 1.
In all strains tested, we failed to detect the following
enzymes of the serine pathway: serine glyoxylate
aminotransferase and ATP-, CoA-dependent malate
lyase, or the key enzyme of the ribulose monophosphate cycle, 3-hexulose phosphate synthase. Hydroxypyruvate reductase activity, both NADH- and
NADPH-dependent was found; however, its level was
similar in methanol- or glucose-grown cells. This
suggests that in these bacteria hydroxypyruvate reductase does not have a function specific to the serine
pathway, but may have a similar metabolic role as
suggested for Pa. denitrificans [5]. It can therefore
be concluded that the serine pathway or the ribulose
monophosphate cycle are not involved in the carbon
assimilation during growth ofAchromobacter sp. 1L,
Pseudomonas sp. 8 and Mycobacterium sp. 50 on
methanol.
Inspection of the occurrence of ribulose bisphosphate carboxylase showed that all three strains
possessed activity of this enzyme. The induction of
ribulose bisphosphate carboxylase in cells growing on
methanol indicates that the route of carbon assimilation is via the Calvin cycle.
Activities of methanol dehydrogenase (NAD- or
PMS-linked) and methanol oxidase could not be
detected in cell-free extracts by the standard assay
procedures. Whole cells, however, oxidized methanol
at high rates in the presence of 2,3,5-triphenyltetrazolium chloride as electron acceptor (600 nmol triphenyl formazan formed min -1 mg of protein-l). It
may be speculated that the enzyme responsible for
primary methanol oxidation is either extremely
labile or requires special assay conditions. Obviously,
the first step of methanol oxidation in these bacteria
remains to be investigated. Two formaldehyde
oxidizing enzymes were detected namely an NADdependent formaldehyde dehydrogenase which
required glutathione for activity and a PMS-linked
enzyme. In all three isolates NAD.dependent formaldehyde dehydrogenase was induced during growth
on methanol. The activity of the PMS-linked enzymes
was somewhat higher in glucose-grown cells as compared to methanol-grown cells. The activity of NADdependent formate dehydrogenase increased tenfold
during growth on methanol as compared to glucosegrown cells. In addition a PMS-linked formate dehy-
242
TABLE 1
Specific activities of enzymes involved in intermediary metabolism of Cl-compounds in extracts of methanol- and glucose-grown
bacteria
Enzyme activity were measured by the spectrophotometric methods indicated in Materials and Methods.
Enzyme
Glucose-6-phosphate dehydrogenase
NAD÷
NADP÷
6-Phosphogluconate dehydrogenase
NAD÷
NADP ÷
Fructose diphosphate aldolase
6-Phosphogluconate dehydrogenase
+Phospho-2-keto-3 -deo xyaldolase
Hexokinase
Pyruvate dehydrogenase N AD÷
Citrate synthase
lsocitrate dehydrogenase
NAD÷
NADP ÷
a-Ketoglutarate dehydrogenase
NAD÷
Malate dehydrogenase
NAD÷
NADP ÷
NADH
NADPH
Isocitrate lyase
Enzyme activities (nmol min -1 mg protein -1 )
Achromobacter 1L
Pseudomonas 8
Mycobacteriurn 50
Methanol
Methanol
Glucose
Methanol
Glucose
Glucose
72
262
80
290
96
230
140
310
70
190
160
240
27
30
70
29
270
120
20
440
89
40
142
130
40
52
60
200
190
120
0
550
15
45
0
1600
17
460
0
330
18
65
0
980
20
420
0
170
40
35
0
270
42
74
0
490
0
970
0
430
0
610
0
270
0
763
18
30
12
20
4
37
65
0
170
50
25
98
0
320
54
40
55
0
200
22
15
68
0
324
97
18
20
0
209
18
4
21
0
430
10
6
drogenase activity has been detected in these bacteria
during growth on methanol.
The enzymic study o f the pathways of intermediary carbon metabolism given in Table 2, has
shown the presence o f a number o f enzymes o f the
glycolytic pathway and oxidative pentose phosphate
pathway namely hexokinase, fructose diphosphate
aldolase, glueose-6-phosphate dehydrogenase and
6-phosphoglyconate dehydrogenase in all three
organisms. The latter two enzymes were active b o t h
with NAD and NADP. It is n o t e w o r t h y that hexokinase, fructose diphosphate aldolase and glucose6-phosphate dehydrogenase (NAD) were present at
higher levels in glucose-grown cells. Aldolase o f
phospho-2-keto-3-deoxygluconate was not found in
any of the isolates. This excludes the possibility o f
the cleavage o f h e x o s e phosphates via the EntnerD o u d o r o f f pathway.
All bacteria tested possessed activities o f pyruvate
dehydrogenase and some enzymes o f the Krebs cycle
and glyoxylate shunt (Table 2). As a rule their levels
were higher in glucose-grown cells. During growth of
these organisms on methanol, the specific activities o f
citrate synthase isocitrate dehydrogenase and a-ketoglutarate dehydrogenase were much lower indicating
a biosynthetic role for the Krebs cycle. It is obvious
that direct oxidation o f methanol via formaldehyde
and formate to CO2 covers the energy requirements
of the organisms.
In view of the above results it seemed relevant to
check whether Achromobacter sp. I L, Pseudomonas
sp. 8 and Mycobacterium sp. 50 could grow autotrophically in an atmosphere o f H2 + CO2 + 02. All
three strains appeared to grow under such conditions
but as in the case o f methanol utilization they
required biotin (30/ag/1). Hence Achromobacter sp.
243
1 L, Pseudomonas sp. 8 and Mycobacterium sp. 50
are typical facultative chemolithotrophs which fix
carbon at the level o f CO2 by the ribulose bisphosphate cycle and are able to generate energy from the
oxidation o f methanol to CO2 via formate. If we
accept the definition o f m e t h y l o t r o p h y given by Colby and Zatman [25] in 1972 the term m e t h y l o t r o p h
does not seem appropriate for these organisms because
they assimilate reduced Cl-compounds autotrophically. However, recently Quayle a n d Ferenci [26]
suggested the term m e t h y l o t r o p h for all organisms
which can grow on reduced Crsubstrates irrespective
o f their mode o f assimilation. Therefore, present-day
relationships between m e t h y l o t r o p h y and a u t o t r o p h y
are not completely clear. The data reported in this
paper support the view o f Quayle and Ferenci [26]
that "these organisms are probably autotrophs by
design and methylotrophs by accident and many
more autotrophs can carry out this type o f metabolism". The recent discoveries by ourselves and others
o f several bacteria belonging to very different genera
which carry out autotrophic growth on methanol
show that this type o f metabolism may not indeed be
rare.
Acknowledgements
We wish to thank Professor E.N. Kondratieva for
kind supply o f bacterial cultures and for attention to
this work. We are grateful to Professor J.R. Quayle
and Dr. J.P. van Dijken for their helpful advice.
References
[ 1 ] Quayle, J.R. (1972) in: Advances in Microbial
Physiology VII (A.H. Rose and D.W. Tempest, Eds.)
Academic Press, New York, pp. 119-203.
[2] Anthony, C. (1975) Sci. Progr. 62,167-206.
[3] Quayle, J.R. and Pfennig, N. (1975) Arch. Microbiol.
102,193-198.
[4] Sahm, H., Cox, R.B. and Quayle, J.R. (1976) J. Gen.
Microbiol. 94,313-322.
[5] Cox, R.B. and Quayle, J.R. (1975) Biochem. J. 150,
569-571.
[6] Chandra, T.S. and Shethna, Y.I. (1976) J. Bacteriol.
131,289-398.
[7] Loginova, N.V., Namsaraev, B.B. and Trotsenko, Y.A.
(1978) Mikrobiologiya (in Russian) 47,168-170.
[8] Str!fim,T., Ferenci, T. and Quayle, J.R. (1974) Biochem. J. 144,465-476.
[9] Troyan, O.S., Kirikova, N.N. and Kondratieva, E.N.
(1975) Sci. Proc. High School, Biol. Sciences (in
Russian) 9,100-104.
[10] Anthony, C. and Zatman, L.J. (1965) Biochem. J. 96,
808-812.
[ 11 ] Tani, Y. Miya, T. and Ogata, K. (1972) Agr. Biol. Chem.
36, 76-83.
[12] Johnson, P.A. and Quayle, J.R. (1964) Biochem. J. 93,
281-290.
[13] Blackmore, M.A. and Quayle, J.R. (1970) Biochem. J.
118,53-59.
[14] Dixon, G.H. and Kornberg, H.L. (1959) Bioehem. J. 72,
3P.
[15] Lawrence, A.J., Kemp, M.B. and Quayle, J.R. (1970)
Biochem. J. 116,631-639.
[16] Watson, B.F. and Dworkin, M. (1968) J. Bacteriol. 96,
1465-1473.
[17] Sibley, J.A. and Lehninger, A.L. (1949) J. Biol. Chem.
177,859-872.
[18] Wood, W.A. (1971) in: Methods in Microbiology, Vol.
6A, (J.R. Norris and D.W. Ribbons, Eds.) Academic
Press, New York, pp. 411-424.
[19] Carls, R.A. and Hanson, R.S. (1971) J. Bacteriol. 106,
848-855.
[20] Ochoa, S., Stem, J.R. and Schneider, M.C. (1951) J.
Biol. Chem. 193,691-702.
[21 ] Kornberg, A. (1955) in: Methods in Enzymology, Vol.
I (S.P. Colowick and N.O. Kaplan, Eds.) Academic
Press, New York, pp. 705-709.
[22] Ochoa, S. (1955) in: Methods in Enzymology, Vol. I
(S.P. Colowiek and N.O. Kaplan, Eds.), Academic Press,
New York, pp. 735-739.
[23] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall,
R.J. (1951) J. Biol. Chem. 193,265-275.
[24] Baumforth,C.W. and Quayle, J.R. (1977) J. Gen. Microbiol. 107, 259-267.
[25] Colby, J. and Zatman, L.J. (1972) Biochem. J. 128,
1373-1376.
[26] Quayle, J.R. and Ferenci, T. (1978) Microbiol. Rev. 42,
251-273.