Journal of General Microbiology (1982), 128, 2927-2935.
Printed in Great Britain
2921
Role of Ribulose-l,5-bisphosphateCarboxylase/Oxygenase in
Methylococcus capsulatus (Bath)
By S . H . S T A N L E Y A N D H . D A L T O N *
Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, U.K.
(Received 2 April 1982; revised 12 June 1982)
Intact cells of the methane-oxidizing organism Methylococcuscapsifatus(Bath) assimilated C 0 2
for several hours in the absence of methane, provided that an alternative energy source such as
hydrogen and/or formate was available. Despite the presence of ribulose bisphosphate
carboxylase and a ribulose monophosphate pathway in this organism, autotrophic growth in the
presence of a suitable energy source could not be demonstrated. Radiolabelling studies
suggested that the C 0 2 was incorporated via C 3 carboxylation and ribulose bisphosphate
carboxylase in the presence of both methane and hydrogen plus formate. However, in the
presence of methane the C 0 2 was further metabolized into sugar phosphates, whereas in the
absence of methane, but with hydrogen plus formate as an energy source, the sugar phosphates
were not labelled to any significant extent. Of the methane-oxidizing bacteria tested, ribulose
bisphosphate carboxylase was found only in M . capsulatus strains (Bath and Foster & Davis).
The ribulose monophosphate pathway in M . capsulatus (Bath) probably uses the keto-deoxy-6phosphogluconate route for the cleavage of fructose 6-phosphate into two C3 molecules rather
than via phosphofructokinase and fructose bisphosphate aldolase which have low activities in
this organism. The function of the ribulose bisphosphate carboxylase may be to provide an
alternative cleavage pathway for the synthesis of 3-phosphoglycerate during growth on
methane.
INTRODUCTION
Methane-utilizing bacteria (methanotrophs) have been classified into two types according to
their pathway of carbon assimilation and internal membrane structure (Whittenbury et al.,
1970). Type I organisms have a ribulose monophosphate (RMP) pathway, bundles of vesicles
throughout the whole cell and a GC content of 50-54% whilst type I1 organisms use the serine
pathway, have membranes around the periphery of the cell and a GC content of 62.5%.
Methylococcus capsulatus (Bath) appeared to be a typical type I organism with bundles of
vesicular membranes and uses the RMP pathway as its main route of carbon assimilation (Strom
et al., 1974; Reed, 1976). However, it also possessed hydroxypyruvate reductase activity (Reed,
1976) and had a GC content of 62.5% (Whittenbury et al., 1975), typical of type I1
methanotrophs. Furthermore, the presence of ribulose bisphosphate carboxylase in M .
capsulatus (Bath) (Taylor, 1977) has led to doubts about the assignment of this organism to the
type I methanotrophs and has therefore been classified as a type X (Whittenbury & Dalton,
1981).
This paper presents further evidence for the role of ribulose bisphosphate carboxylase in the
overall metabolism of M . capsulatus (Bath).
Abbreviations:DHAP, dihydroxy acetone phosphate ; FBP, fructose bisphosphate; F6P, fructose 6-phosphate ;
GAP, glyceraldehyde 3-phosphate ; G6P, glucose 6-phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate;
NMS, nitrate mineral salts; PEP, phosphoenol pyruvate ;6PG, 6-phosphogluconate; 3PGA, 3-phosphoglycerate ;
RuBP, ribulose bisphosphate; RUMP, ribulose 5-phosphate ; RuBPCase, ribulose bisphosphate carboxylase.
0022-1287/82/O001-0508 $02.00 0 1982 SGM
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S . H . STANLEY A N D H . D A L T O N
METHODS
Growth of organisms. Methylococcus cupsulutus (Bath) (Whittenbury et ul., 1970)was grown in continuous culture
in a 5 1 fermenter (LKB) on nitrate mineral salts (NMS) medium (Dalton & Whittenbury, 1976). The fermenter
was operated under both oxygen- and methane-limited conditions with dissolved oxygen control on methanelimited cultures when necessary. Temperature and pH value of the culture were maintained at 45 "C and 643,
respectively. Unless otherwise stated the chemostat was operated at a dilution rate of approximately 0.1 h-l and a
cell density of 1 mg dry wt ml-l. All other organisms were grown at 30 "C either in continuous culture or in 3 1
fermenters as batch cultures.
Autotrophic growth of M . cupsulutus (Bath) was attempted in 250 ml conical flasks or in 25 ml Universal bottles
stoppered with rubber serum caps (Suba Seal, W. H. Freeman, Barnsley, U.K.). Gases (methane, CO,, oxygen
and hydrogen) were added at concentrations of 1-20% (v/v) and soluble substrates up to 10 mM. The experiments
with hydrogen and/or formate were also undertaken in a closed batch system in which the gas phase was
continuously recirculated, by pumping, through the culture, gas sampling valves and a manometer device for
maintaining atmospheric pressure. Samples of the gas phase (0-5 ml) were periodically removed via the gas
sampling valves and analysed on porapak R and molecular sieve in a Pye 104 gas chromatograph equipped with a
katharometer.
Preparation of cell extracts. Cells were harvested by centrifugation at 10000g for 10 min, washed once with, and
resuspended in, 20 mM-Tris/HCl, pH 7.0 containing 10 mM-MgC1,. Crude cell extracts were prepared by two
passages of the suspension through a pre-cooled French pressure cell (American Instrument Co., Maryland,
U S A . ) at 137 MPa. Unbroken cells and debris were removed by centrifugation at lOOOOg for 10 min and the
supernatant was further centrifuged at 32000 g for 30 min to yield soluble and particulate fractions.
Enzyme ussuys. RuBPCase [3-phospho-~-glyceratecarboxy-lyase (dimerizing); EC 4.1.1.391 was assayed by
the RuBP-dependent incorporation of NaH14C03 into acid stable products (Taylor, 1977). G6P isomerase (Dglucose-6-phosphate ketol-isomerase; EC 5.3.1 .9) was assayed by measuring the formation of G6P from F6P
EC 1.1.1.49) was assayed by the
(Noltmann, 1966).G6P dehydrogenase (~-glucose-6-phosphateoxidoreductase;
method of Kuby & Noltmann (1966) except that Tris/HCl was used instead of glycylglycine. 6PG dehydrogenase
[6-phospho-~-gluconate oxidoreductase (decarboxylating); EC 1.1.1.441 was assayed by the method of
Pontremoli & Grazi (1966) except that TrisfHCl buffer was used. 6PG dehydratase (6-phospho-~-gluconate
~-glyceraldehyde-3hydrolyase; EC 4.2.1 .12) and KDPG aldolase (6-phospho-2-keto-3-deoxy-~-gluconate
phosphate-lyase; EC 4.1.2.14) were assayed by measuring the formation of GAP from 6PG (Wood, 1971).
Phosphofructokinase (ATP :D-fructose-&phosphate 1-phosphotransferase; EC 2.7.1 . 11) was assayed by using
the linked enzyme method of Ling et ul. (1966). FBP aldolase (D-fructose-l,6-bisphosphate~-glyceraldehyde-3phosphate lyase; EC 4.1.2.13) was assayed by using the linked enzyme method of Rutter & Hunsley (1966). The
combined activities of 3PGA kinase (ATP :3-phospho-~-glycerate1-phosphotransferase; EC 2.7.2.3) and GAP
:NAD oxidoreductase (phosphorylating); EC 1.2.1.12] were
dehydrogenase [~-glyceraldehyde-3-phosphate
determined by measuring the rate of NADH oxidation (spectrophotometrically at 340 nm) in the presence of
3PGA and ATP, corrected for the rate of NADH oxidation in the following controls: 1, no ATP; 2, no 3PGA; 3,
only NADH and extract. The assay mixture contained 50 mM-Tris/HCl pH 7.5,2 mM-MgCl,, 1 mM-ATP, 1 mM3PGA, 0.15 mM-NADH and extract. Formate dehydrogenase (formate : NAD+ oxidoreductase; EC 1.2.1.2) was
assayed by the method of Quayle (1966). PEP synthetase was assayed by measuring pyruvate disappearance in the
presence of ATP or pyruvate appearance with PEP and AMP using the methods of Cooper & Kornberg (1969).
Enzyme assays were performed at 45 "C for extracts of M. cupsulatus (Bath) except for linked enzyme procedures
which were assayed at 35 "C. All enzyme assays for the other methanotrophs were performed at 30 "C.
Radiotracer studies. These were carried out by the methods of Taylor et ul. (1981) except that the radiolabel
uptake experiment was performed as follows: a suspension (1 ml; 1 mg dry wt ml-l) of methane-grown cells of M.
cupsulutus (Bath) was taken from an oxygen-limited continuous culture and incubated in a sealed flask containing
either hydrogen (20%, v/v) and potassium formate (10 m~ final concentration), or methane (20%, v/v). After
5 min incubation 20 pCi NaHI4CO3 (740 GBq) was added and after 10 s 4 ml hot ethanol was added to stop all
metabolic processes and to extract the pool metabolites from the cells. This procedure was repeated but with the
exposure time to the label increased to 20,30 and 45 s. The pool metabolites were extracted and separated by twodimensional paper chromatography.
RESULTS A N D DISCUSSION
C 0 2fixation by whole cells of M . capsulatus (Bath)
Taylor et a/. (198 1) reported that C 0 2fixation in whole cells of M . capsulatus (Bath) required
the addition of methane, and that no significant C 0 2 incorporation occurred in the absence of
this substrate. They suggested that the methane provided energy for C 0 2 fixation and that
perhaps other oxidizable substrates (formate, hydrogen etc.) may also drive this incorporation.
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Methylococcus capsulatus RuBP
Table 1. CO, incorporation rates in cell suspensions of M . capsulatus (Bath)
+
+
Scintillation vials (30 ml capacity) containing the following additions 1 ml COz 1 pCi NaH14C03
in 0.9 ml NMS were inoculated with an actively growing culture of M .capsulatus (Bath) (0-1 ml of 2 mg
dry wt cells ml-I) and incubated for 1 h with shaking at 45 "C.The incubation was stopped by the
addition of 100 p1 12 M-fOrmiC acid, the contents heated to dryness and the remaining acid-stable I4C
was determined.
Addition
None
CH4 (5%)
H2 (5%)
HCOOK ( l o r n )
H2 (5%) +
HCOOK (10 mM)
Methanol (50 mM)
NH4Cl (10 mM)
COz incorporation rate
[nmol COz h-' (mg dry weight cells)-l]
30
410
195
250
740
215
30
To test this hypothesis, flasks containing 4C-labelled bicarbonate and various energy sources
were inoculated with an actively growing culture of M . capsulatus (Bath). The results (Table 1)
confirmed that only low levels of C 0 2 were incorporated in the absence of an energy source,
whereas in the presence of oxidizable energy sources the rates of CO, uptake were enhanced at
least sixfold. In other experiments (results not shown) it was observed that CO, incorporation
could be driven by hydrogen and/or formate for at least a 7 h period suggesting that one or
several of the following possibilities occurred : 1 , a fully operative Calvin cycle was present; 2,
there were sufficient endogenous carbon skeletons produced to enable known heterotrophic
carboxylations to occur; 3, that RuMP was generated from known reserve polymers such as
polyglucose (Linton & Cripps, 1978) thereby permitting CO, fixation to occur via
phosphoribulokinase and RuBPCase. The possible contribution of these various routes of CO,
incorporation was therefore investigated.
Fate of incorporated HI4Co3 in M . capsulatus (Bath)
The fate of COz in the presence of methane or hydrogen and formate was investigated by
following the early time course of labelling of pool metabolites from Hl4CO3.The results from
the incubation with methane (Table 2, part b) confirmed the findings of Taylor et a f .(1981) that
both C3 carboxylation and RuBPCase were involved in C 0 2 incorporation in this organism.
The early label in malate and aspartate was indicative of C3 carboxylation forming malate or
oxaloacetate. The label appearing in 3PGA and sugar phosphates demonstrated that the
RuBPCase was active and that the 3PGA produced may be further metabolized by enzymes of
the RuMP pathway. Results with hydrogen and formate (Table 2, part a ) showed a similar
pattern with evidence of both C3 and C5 carboxylations occurring but with one major
difference: in the presence of methane about 30% of the label appeared in four spots
corresponding to sugar phosphates on the chromatograms, whereas with hydrogen and formate
a maximum of only 17% of the label appeared in two spots within the sugar phosphate region,
close to the 3PGA spot, of which only one spot may be the same as those labelled in the presence
of methane. This lack of label in the sugar phosphates but heavy label in the region
corresponding to 3PGA with hydrogen and formate suggests that the RuBPCase is functional
under these circumstances but that the 3PGA produced may only be metabolized to a limited
extent by a Calvin or RuMP pathway. If this organism is to grow autotrophically then
presumably a functional Calvin cycle will be required, of which most of the necessary enzymes
are already present as components of the RuMP pathway. Furthermore an energy source will
also be necessary; this can be supplied as hydrogen and/or formate, which are known to drive
the energy-requiring process of acetylene reduction in this organism (Dalton & W hittenbury,
1978). Formate is oxidized to CO, in this organism by an NAD+-dependent formate
dehydrogenase with activities between 100 and 400 nmol min-I (mg protein)-': However, the
labelling data indicate that a Calvin cycle may not operate in M . capsulatus (Bath) to any
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S. H . STANLEY A N D H . DALTON
Table 2. Percentage incorporation of I4CO2 into pool metabolites of M . capsulatus (Bath) at
45 "C
(a) In the presence of 20% (viv) H 2 and 10 mM-pOtaSSiUm formate
Radioactivity in metabolite
(% of total in sample)
Metabolite
10
3PGA
Sugar
phosphates
Aspartate
Malate
PEP
Glutamate
Citrate
46-1
0
0
14.2
12-3
13.0
6.4
5.9
Sample time (s):
20
30
33.6
7.1
10.2
13.4
9.0
10.1
6.2
4.8
31.8
9.4
6-9
13.1
7.4
13.6
3.2
5.7
45
35.8
9.2
0
8.4
6.2
8-1
5.1
5.5
(6) In the presence of 20% (v/v) CH,
Radioactivity in metabolite
(% of total in sample)
Metabolite
3PGA
Sugar
phosphates
Aspartate
Malate
PEP
Glutamate
li
3
10
16.4
7.2
2.9
10.4
4.5
26.3
12.4
8.3
2-7
Sample time (s):
20
30
18.2
12.8
7*1
11.5
5.5
22.3
11.4
5.9
-
45
22.9
8.7
12.2
11-1
9.8
12.4
2.3
21.9
11.0
2.7
7.0
4.9
20-7
11.9
3.8
7.8
} 18.3
* This sugar phosphate spot may be similar to sugar phosphate spot no. 2 in the CH, experiment.
significant extent. It should be emphasized that these experiments were done with cells taken
from cultures growing on methane and that they may not have had time to adapt to the
autotrophic conditions presented to them. To determine if this were true the possibility of
autotrophic growth by this organism was investigated.
Attempts to grow M . capsulatus (Bath) autotrophicully
Specific activities of RuBPCase in crude cell extracts which were sufficient for autotrophic
growth in a number of organisms varied from 2-8-40nmolmin-' (mg protein)-' (Malik &
Schlegel, 1981).On the basis of this criterion activities of RuBPCase observed in cell extracts of
M . cupsulutus (Bath) [10-40 nmol min-' (mg protein)-'] and phosphoribulokinase [3.3 nmol
min-' (mg protein)-'] (Taylor, 1977) should be high enough to support autotrophic growth of
this organism. Furthermore it is possible that the carboxylase activity was repressed under
methane-excess conditions (oxygen limitation was the usual cultural condition), whereas under
methane-limited conditions (carbon-limited but with 2-5 % C 0 2 in the gas phase) this activity
may be derepressed. To investigate whether this may occur, M . capsulatus (Bath) was
established in continuous culture and the activity of the RuBPCase in cell extracts was
determined under both oxygen- and methane-limited conditions. However no consistent change
in the activity of this enzyme was detected, even with the addition of 10 mwformate to the
media.
Since M. capsulatus (Bath) could use hydrogen and formate as energy sources to drive C 0 2
fixation, attempts were made to see if this organism could be persuaded to grow under
autotrophic conditions. Flasks containing hydrogen and/or formate and C 0 2 were inoculated
with methane-grown cells. No growth was detected in these flasks even after several weeks,
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Methylococcus capsulatus RuBP
whereas growth did occur overnight in control flasks containing methane. These experiments
were repeated with supplements of glycine, serine, pyruvate or malate at concentrations of
10 mM; again no growth occurred in the absence of methane. The pH value rose quite rapidly
when M . capsulatus (Bath) oxidized potassium formate, for example, a culture with a cell density
of 0.1 mg dry wt ml-l raised the pH value from 6.9 to 7.3 in 5 h in the presence of 10 mMpotassium formate. Since this rise in pH value may inhibit growth a closed batch culture system
was established on NMS containing 10 mM-potassium formate in which 0.1 M-formic acid was
added to maintain the pH value at 6.8. This batch growth system was inoculated with methanegrown cells. For a period of 3 d the formate was oxidized with concomitant C 0 2 production,
oxygen utilization and addition of formic acid to control the pH value but no growth could be
demonstrated either by optical means or by using a Coulter counter to estimate the number of
cells directly. Similar experiments using C02/hydrogenor formate/CO,/hydrogen mixtures as
carbon and energy sources were undertaken but again no growth was detected. Since this
organism does not appear to grow autotrophically one can ask the question, ‘what function does
the RuBPCase fulfil in the metabolism of this organism?’.
Possible roles of RuBPCase in M . capsulatus (Bath)
Investigation of the presence of RuBPCase in methanotrophs indicated that this enzyme was
present only in M . capsulatus strains and possibly present to a very limited extent in
Methylomonas methanica (strain SI) (Table 3). Therefore, RuBPCase is not necessary for growth
on methane by all type I methanotrophs. The presence of this enzyme in M . capsulatus strains
may provide these organisms with some advantage over other type I organisms or perhaps a
lesion in their metabolism necessitates its activity. It has been noticed on several occasions that
M . capsulatus (Bath) failed to grow on methane in shake flasks if C 0 2 (0.3-2%, v/v) was not
included in the gas phase. The requirement for C 0 2 may reflect a necessary role of the
RuBPCase, although C 3 carboxylations will also be necessary for growth. One possibility is that
the RuBPCase provides an alternative C, cleavage route in the RUMPpathway (Taylor, 1979;
Table 3 . RuBPCase and FBP aldolase activities in extracts of methane-grown methanotrophs
Enzyme activity
[nmol min-I (mg protein)-’]
Type
Organism
RuBPCase
FBP aldolase
X
X
I
I
I
I
I
I
I1
I1
I1
Methylococcus capsulatus (Bath)
Methylococcus capsulatus (Foster & Davis)
Methylomonas methanica SI
Methylomonas methanica PM
Methylomonas methanica A4
Methylomonas agile (A20)
Methylomonas albus (BG8)
Methylobacter capsulatus (Y)
Methylosinus trichosporium (OB3b)
Methylosinus trichosporium (OBBP)
Methylosinus sporium
10-40
0-5
4-1*
0-0.2
0
O*
O*
0
0
O*
O*
O*
107
8t
45
34
27
-
30
-
-, Assays not done. * Results of Taylor (1979). 7 Result of Strom et a f . (1974).
Table 4. Energy balance of diflerent cleavage schemes
These energy balances are for the formation of 1 mol pyruvate from 3 mol formaldehyde.
Scheme
KDPG aldolase
FBP aldolase
RuBPCase
NAD(P)H
ATP
+1
+1
+1
+1
-1
0
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S. H . STANLEY A N D H . DALTON
Quayle, 1979).These routes generate two C 3 fragments, one for assimilation and the other for reentry into the rearrangement sequences of the RuMP pathway via GAP. The three possible c6
cleavage routes are represented in Fig. 1. The phosphoribulokinase/RuBPCase variant
providing 2 mol3PGA, one of which can be converted to GAP for rearrangement and one for
assimilation via the C 3 components of the glycolytic sequence. This cleavage pathway is
compared with the other two pathways in terms of NAD(P)H/ATP generation or consumption,
starting with 3 mol formaldehyde to form 1 mol pyruvate for assimilation (Table 4). The
cleavage pathway using RuBPCase is less favourable in terms of ATP balance compared to the
other two routes.
Methylococcus methanica and M . capsulatus (Foster & Davis) possess enzymes of both the FBP
and KDPG aldolase variants of the c6 cleavage pathway (Strom el al., 1974). It is not known if
one or both pathways operate in uiuo but the FBP aldolase pathway is energetically more
favourable. The levels of these enzymes were investigated in M . capsulatus (Bath) and the results
presented in Table 5 . The activities of phosphofructokinase and FBP aldolase were difficult to
assay because of their low rates compared to the NADH oxidase activities in these extracts [4060 nmol min-’ (mg protein)-’]. A further complication of the phosphofructokinase assay was
that F6P can be metabolized to G6P which in turn can be oxidized by the NAD(P)-linked G6P
dehydrogenase.
HCHO
.RUMP
A HuMP
HCHO ATP-”/PRK
1 I
I
NADH
RuBPCase
‘
F6P -G6P
RuBP
G y
PGI
+2Ru[P
+2HCHO
C O ~ 3- PGA
~ 3 ~ F ~ T ~
FBP
6 PG
2 F6P
6 PGDHy
1
DPGA
NADH
7
Rearrangement
reactions of the
RuMP pathway
+1
4
4
Pyruvate
3PGA
Pyruvate
Pyruvate
+
I
GAP
GAP
GAP
0
Fig. 1. Alternative routes of C , cleavage of the RuMP pathway. 1, Phosphoribulokinase/RuBPCase
route. 2, FBP aldolase route. 3, KDPG aldolase route. PRK, phosphoribulokinase; PFK,
phosphofructokinase; FBPA, fructose bisphosphate aldolase ; PGI, glucose 6-phosphate isomerase ;
G6PDH, glucose 6-phosphate dehydrogenase, 6PGDHy, 6-phosphogluconate dehydratase ; KDPGA,
keto-deoxy-phosphogluconatealdolase ; DPGA, 1,3-diphosphogIycerate; HuMP hexulose monophosphate and HCHO formaldehyde.
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Methylococcus capsulatus RuBP
Table 5. Enzyme activities in extracts of M . capsulatus (Bath)
Activity
[nmol min-' (mg protein)-']
Enzyme
Phosphoglucose isomerase
Glucose-6-P-dehydrogenase (N ADP)
Glucose-6-P-dehydrogenase (N AD)
6-P-Gluconate dehydratase
Keto-deoxy-6-P-gluconate aldolase
P hosphofructokinase
Fructose bisphosphate aldolase
6-Phosphogluconate dehydrogenase (NADP)
6-Phosphogluconate dehydrogenase ( N AD)
3-Phosphoglycerate kinase
Glyceraldehyde phosphate dehydrogenase
Ribulose bisphosphate carboxylase
Formate dehydrogenase (NAD)
ND,
186-208
1&32
28-56
} 38-44
0-6
0-5
39-53
ND
} 35-55
10 4 0
100400
Not detected.
These results indicated that M . capsulatus (Bath) has all the enzymes necessary for the KDPG
aldolase route of C6 cleavage and possibly low activities of the FBP aldolase route. The branch
point for the two cleavage pathways occurs at the level of F6P (Fig. 1) which can be converted to
G6P, for the KDPG aldolase pathway, or to FBP, for the FBP aldolase pathway. The G6P
isomerase which converts F6P to G6P, was over 30 times more active than the
phosphofructokinase which converts F6P to FBP. This imbalance of activities may lead to most
of the F6P being metabolized via the KDPG aldolase pathway of hexose cleavage. Furthermore
F6P was also a substrate for the transaldolase and transketolase of the rearrangement reactions
of the RuMP pathway, so this compound is even less likely to serve as a substrate for the low
levels of phosphofructokinase detected in this organism.
The products of the KDPG aldolase pathway were GAP and pyruvate, presumably the GAP
is fed back into the rearrangement reactions of the RuMP pathway and the pyruvate is further
used for cell synthesis. The C3 components of glycolysis from DHAP to PEP could be made via
the FBP aldolase pathway (Fig. 1) or from pyruvate via pyruvate carboxylase/PEP
carboxykinase or PEP synthetase to provide PEP. As indicated above it is unlikely that the FBP
aldolase pathway functions to any significant extent. Furthermore the activities of the pyruvate
carboxylase and PEP carboxykinase are less than 1 nmol rnin-l (mg protein)-' (Taylor, 1979)
and although ATP occasionally stimulated pyruvate disappearance no PEP could be detected as
a product and no activity for the reverse reaction of the PEP synthetase could be found.
Therefore it can be concluded that the low or non-existent activities of these possible routes may
lead to a shortage of C3 components which could be supplied by the RuBPCase route of C6
cleavage through the generation of two units of 3PGA. One unit of 3PGA being used for
synthesis of GAP to replenish the rearrangement reactions of the RuMP pathway and the other
unit for synthesis of the C3 components of glycolysis between DHAP and PEP. The enzymes for
conversion of 3PGA to GAP were present in extracts of this organism (Table 5).
Other type I methanotrophs which do not possess RuBPCase must have either the FBP
aldolase cleavage pathway or another route of C3 synthesis. When tested it was found that all the
type I methanotrophs had significant levels of FBP aldolase compared to that found in M .
capsulatus (Bath) (Table 3). Strom et al. (1974) found low levels of FBP aldolase in M . capsulatus
(Foster & Davis) and as Taylor (1979) demonstrated that this strain also had RuBPCase it is
tempting to suggest that the presence of this enzyme is related to the low activity of the FBP
aldolase cleavage route.
The lack of or low activity of FBP aldolase may explain why we have been unable to
demonstrate autotrophic growth of M . capsulatus (Bath). FBP aldolase is a necessary enzyme of
the Calvin cycle, forming FBP from two C3 units provided by the RuBPCase. If this organism
cannot synthesize sugar phosphates from 3PGA then the Calvin cycle will not function. Low
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S. H . STANLEY A N D H . DALTON
activities of the FBP aldolase may not produce enough FBP to maintain the cycle. This
hypothesis could also explain the radiolabelling data. In the presence of methane the early label
from H14C03 which appeared in 3PGA could be incorporated into the RuMP pathway via
GAP in the transketolase reaction. This was demonstrated by early label appearing in several
sugar phosphates. In the presence of hydrogen and formate however, only one of these sugar
phosphates may have been labelled demonstrating that the RuMP pathway was not functional
(due to lack of formaldehyde to drive this pathway) and that a Calvin cycle could not turn
because little or no FBP could be formed for the condensation of GAP and DHAP.
The RuBPCase may also play a role in the overall energy balance of the cell. Methylococcus
capsulatus (Bath) must have some control over the fate of formaldehyde, which can be
assimilated or oxidized to C02. The possible control points occur at two levels in the metabolic
pathways of this organism either: 1, at the level of formaldehyde and its subsequent oxidation to
C 0 2 or condensation with RuMP for assimilation or 2, at the level of 6PG which can be
converted to KDPG for assimilation or oxidized to C 0 2 and RuMP by the 6PG dehydrogenase
present in this organism. Whether formaldehyde is oxidized to C 0 2 by the NAD-dependent
formaldehyde dehydrogenase (Stirling & Dalton, 1978)/formate dehydrogenase and/or the
cyclic pathway (G-6-P dehydrogenase/G-P-G dehydrogenase) in vivo is not known. If the control
over oxidation or assimilation is not precise then any excess C 0 2 production will result in the
loss of carbon from the system and wasteful energy production. The alternative cleavage
pathway using the RuBPCase may provide a means of utilizing this wasteful energy production
and recover some of the lost carbon.
We thank Mr S. J. Pilkington for his excellent technical assistance and Dr S. C. Taylor for the RuBPCase assays
on the methanotrophs. This work was funded through an SERC research grant to H. D.
REFERENCES
COOPER,R. A. & KORNBERG,
H. L. (1969). Phosphoenolpyruvate synthetase. Methods in Enzymology 13,
309-3 14.
DALTON,
H. & WHITTENBURY,
R. (1976). The acetylene
reduction technique as an assay for the nitrogenase
activity in the methane oxidizing bacterium
Methylococcus capsulatus strain Bath. Archives of
Microbiology 109, 147-1 5 1.
KUBY,S. A. & NOLTMANN,
E. A. (1966). Glucose 6phosphate dehydrogenase (crystalline) from brewer's yeast. Methods in Enzymology 9, 116-125.
LING,K. H., PAETKAU,
V., MARCUS,
F. & LARDY,H.A.
(1966). Phosphofructokinase. Methods in Enzymology 9, 425-429.
LINTON,
J. D. & CRIPPS,R. E. (1978). The Occurrence
and identification of intracellular polyglucose storage granules in Methylococcus NCIB 11083 grown in
chemostat cultures on methane. Archiues of Microbiology 117, 4148.
MALIK,K. A. & SCHLEGEL,
H. G. (1981). Chemolithoautotrophic growth of bacteria able to grow under
N 2-fixing conditions. FEMS Microbiology Letters
11, 63-67.
NOLTMANN,
E. A. (1966). Phosphoglucose isomerase.
Methods in Enzymology 9, 557-565.
PONTREMOLI,
S. & GRAZI, E. (1966). 6-Phosphogluconate dehydrogenase. Methods in Enzyrnology 9,
137- 142.
QUAYLE,
J . R. (1966). Formate dehydrogenase. Methods in Enzymology 9, 360-364.
QUAYLE,
J. R. (1979). Microbial assimilation of C,
compounds. Biochemical Society Transactions 8, 110.
REED,H. L. (1976). A study of certain unusual biochemical and physiological properties of obligate
methane utilizing bacteria. Ph.D. thesis, University of
Warwick.
RUTTER,W. J. & HUNSLEY,
J. R. (1966). Fructose
diphosphate aldolase. Methods in Enzymology 9,
480-486.
STIRLING,D. I. & DALTON,H. (1978). Purification
and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus
capsulatus (Bath). Journal of General Microbiology
107, 19-29.
STROM,T., FERENCI, T. & QUAYLE,
J. R. (1974). The
carbon assimilation pathways of Methylococcus
capsulatus, Pseudomonas methanica and Methylosinus
trichosporium (OB3b) during growth on methane.
Biochemical Journal 144, 465476.
TAYLOR,S. C. (1977). Evidence for the presence of
ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase in Methylococcus capsulatus (Bath).
FEMS Microbiology Letters 2, 305-307.
TAYLOR,S. C. (1979). Ribulose 1,5-bisphosphate
carboxylase and carbon dioxide fixation in Rhodomicrobium vannielii (RM5) and Methylococcus capsulatus (Bath). Ph.D. thesis, University of Warwick.
TAYLOR,
S. C., DALTON,H. & Dow, C. S. (1981).
Ri bulose-l,5-bisphosphate carboxylase/oxygenase
and carbon assimilation in Methylococcus capsulatus
(Bath). Journal of General Microbiology 122, 89-94.
WHITTENBURY,
R. & DALTON,
H. (1981). The methylotrophic bacteria. In The Prokaryotes, pp. 894-902.
Edited by M. P. Starr, H. Stolp, H. G. Triiper, A.
Balows & H. G. Schlegel. Berlin : Springer-Verlag.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:05:46
Met hy lococcus capsula tus RuBP
WHITTENBURY,
R., PHILLIPS,
K. C. & WILKINSON,
J . F.
(1970). Enrichment, isolation and some properties of
methane-utilizing bacteria. Journal of General Microbiology 61, 205-218.
WHITTENBURY,
R., DALTON,H., ECCLESTON,
M. &
REED,H. L. (1975). The different types of methane
oxidizing bacteria and some of their more unusual
properties. In Microbial Growth on C 1 Compounds:
2935
Proceedings of the International Symposium on Microbial Growth on C,-Compounds, pp. 1-9. Edited by G.
Teriu, Tokyo. Osaka: Society of Fermentation Technology.
WOOD, W. A. (1971). Assays of enzymes representative
of metabolic pathways VI 2-keto-3-deoxy-6phosphogluconic aldolase. Methods in Microbiology
6A, 411424.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:05:46