Download The acetyl-CoA pathway of autotrophic growth

FEMS Microbiology Reviews 39 (1986) 345-362
Published by Elsevier
345
FER 00042
The acetyl-CoA pathway of autotrophic growth
(CO dehydrogenase; acetogenesis, methanogenesis; sulfate-reducers)
H a r l a n d G. W o o d , Steve W. R a g s d a l e a n d E w a P e z a c k a
Department of Biochemisto', Case Western Resen'e Unit'ersitv, Clet,eland, OH 44106. U.S.A.
Received 26 FebruaD' 1986
Accepted 8 April 1986
1. S U M M A R Y
The most direct conceivable route for synthesis
of multicarbon compounds from CO: is to join
two molecules of CO 2 together to make a 2-carbon
compound and then polymerize the 2-carbon compound or add CO 2 successively to the 2-carbon
compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum, grows autotrophically by such a process. The mechanism
involves the reduction of one molecule of CO 2 to a
methyl group and then its combination with a
second molecule of CO2 and CoA to form acetylCoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other
bacteria that grow with CO 2 and H 2 as the source
of carbon and energy. This group includes bacteria
which, like C. thermoaceticum, produce acetate as
a major end product and are called acetogens or
acetogenic bacteria. It also includes the methaneproducing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway
occurs in other bacteria by a mechanism that is
the same or similar to that found in C. thermoaceticum. For this purpose, the mechanism of the
acetyl-CoA pathway, as found in C. thermoaceticum, is described and hypothetical mecha-
nims for other organisms are presented based on
the acetyl-CoA pathway of C. thermoaceticum.
The available data have been reviewed to determine if the hypothetical schemes are in accord
with presently known facts. We conclude that the
formation of acetyl-CoA by other acetogens, the
methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C.
therrnoaceticum.
2. I N T R O D U C T I O N
Our definition of an autotrophic organism is an
organism that uses CO 2 (or CO) as the source of
carbon for growth. We are in agreement with
Schlegel [21 and others [3,4] that organisms should
be included as autotrophs even though they do not
have the ability to synthesize from CO 2 certain
vitamins or cofactors which are recycled in
metabolism. We are not in agreement with Whittenbury and Kelly [5] who have expanded the
definition of autotrophy to include all organisms
which utilize organic one-carbon compounds, such
as formate, methanol, methyl amines or methane
as the source of carbon.
The distinctive feature of any autotrophic pathway is the mechanism by which CO 2 is utilized for
the total synthesis of an organic compound from
which the succeeding anabolic reactions proceed.
0168-6445/86/$06.30 ':'31986 Federation of European Microbiological Societies
346
For the most part, following this initial synthesis,
the other mechanisms of CO2 fixation, the synthesis of fatty acids, carbohydrates, proteins and
nucleic acids are similar to those used by organisms
that require organic carbon for growth.
Prior to the discovery of the acetyl-CoA pathway, there were only two pathways known for
autotrophic growth with CO2, as the source of
carbon; they being the reductive pentose cycle
which was discovered by Calvin and his coworkers, and is described in all biochemical texts,
and the reductive tricarboxylic acid cycle. The
Calvin cycle is employed by the majority of autotrophic forms including photosynthetic as well as
chemosynthetic autotrophs. The distinctive enzymes of the Calvin cycle are phosphoribulose
kinase and ribulose-l,5-diphosphate carboxylase.
The latter enzyme generates 3-phosphoglycerate
from the ribulose-l,5-bisP by CO2 fixation and
thus provides the starting material for the anabolic
reactions of these autotrophs.
The reductive tricarboxylic acid cycle occurs by
reverse of the Krebs cycle. There are four CO 2
fixation reactions: acetyl-CoA to pyruvate, pyruvate to oxalacetate, succinyI-CoA to e~-ketoglutarate and a-ketoglutarate to isocitrate. Citrate
formed from the isocitrate is then cleaved by a
citrate lyase to oxalacetate and acetyl-CoA and
the cycle is repeated. In this case, oxalacetate or
acetyl-CoA is the starting material for the anabolic
reactions. This pathway has only been shown to
occur with a limited number of green sulfur
bacteria [6-8].
3. T H E ACETYL-CoA PATHWAY OF ACETOGENS
3. l. The acetyl-CoA pathway of C. thermoaceticum
Surprisingly, the discovery of the acetyl-CoA
pathway came about through study of the metabolism of a heterotroph, C. thermoaceticum. This
organism ferments glucose with the formation of
about 3 tool of acetate from a mol of glucose. In
1945, Barker and Kamen [9], using t4co2, showed
that CO 2 was converted to both the methyl and
carboxyl positions of acetate. The developments
since that time which have led to recognition that
this is an autotrophic pathway have been reviewed
recently [ 10-13].
An outline of this autotrophic pathway is presented in Fig. 1. The scheme has passed through
various modifications which will not be reviewed
nor will the relationship of the scheme to pyruvate
[14] and heterotrophic metabolism be reviewed.
Before considering the scheme in Fig. 1, a few
comments are required concerning the enzyme.
CO dehydrogenase, which we will abbreviate as
CO-DH and represent by ~
with three binding sites X, Y, Z. in Fig. 1. Yagi [15] discovered
this enzyme in 1958 and since then it has been
found in many anaerobic and aerobic bacteria (see
[16] and [17] for references). The enzyme catalyzes
the following reaction with methylviologen as an
artificial electron acceptor and has been purified
from both anaerobic and aerobic bacteria.
CO + H : O ~ CO2 + 2H ' + 2e
(1)
The C O - D H from anaerobes contains Ni and
Fe-sulfur centers [18], whereas that from aerobes
contains Mo instead of Ni [19]. The importance of
this enzyme in the metabolism of C. thermoaceticum became apparent when it was found that
CO could replace pyruvate as a source of the
carbonyl group of acetyl-CoA and that acetyl-CoA
was synthesized from CH3THF, CoASH and CO
[20]. At that time. it was considered that the role
of CO-DH is to catalyze the conversion of CO or
CO 2 to a C~ intermediate that is converted to the
carbonyl group of acetyl-CoA. When it was discovered that CO-DH per se catalyzes an exchange
of CO with CH 3 [14C]OSCoA, we were prompted
to expand the concepts of its function [21]. It
became clear that C O - D H catalyzes the final step
in the synthesis of acetyl-CoA. The exchange involves the cleavage of the C - C and the C - S bonds
of acetyl-CoA and then equilibration of the CO
from the carbonyl group with the CO of the gas
phase. The exchange is illustrated in Eqn. 2 below
in which [1-14C]acetyl-CoA is used and the conversion of a4C to CO is a measure of the exchange.
CH3
C-O +
I
SCoA
~ [y_14CO ~
+ 1[,
LZ-SCoA
LZ-SCoA
12CO
(2)
347
The CO 2 that gives rise to the methyl group of
acetate is introduced by reduction of CO 2 to formate which is converted to formyltetrahydrofolate
and reduced to CH3THF (reaction 2 of the
scheme). The enzymes for this conversion have
been isolated and characterized by Ljungdahl and
co-workers [10,23]. This portion of the scheme is
on a firm basis.
The efforts of our laboratory have been on the
remainder of the scheme. Four enzymes and ferredoxin are required for the conversion of
CH3THF, CO and CoASH to acetyl-CoA and
they have been purified [18,24-26]. The methyltransferase [24] (CH3Tr) catalyzes the transfer of
the methyl from CH3THF to the corrinoid enzyme
([Co]E) [25] (3 of the scheme). The methyl from
CH 3 [Co] E is transferred to the X site of CO-DH
(5 of the scheme). Then it is proposed an acetyi
group is formed at the Y site (6 of the scheme).
This suggestion is based on the observation that
[14C]acetate is formed with [~4C]methyl corrinoid
enzyme, CO and C O - D H [26]. It is postulated the
Since no external acceptors are aded, the acceptors for the methyl, carbonyi and SCoA groups
must be on the CO-DH and are represented by X,
Y. Z in the above reaction. Clearly, for the reaction to be reversible, CO-DH must catalyze the
synthesis of acetyl-CoA from its component parts.
Now we will consider the overall scheme of Fig.
1. H 2 is required as a source of energy and
electrons when the bacteria grow with CO 2 as the
source of carbon. Hydrogenase with H 2 produces
these electrons [22] (reactions 1 and la of the
scheme). Thus, when CO-DH is coupled with hydrogenase, CO 2 is reduced to the C~ precursor of
the carbonyl group of acetyl-CoA (4 of Fig. 1 as
Y-CO). With CO as the substrate, the CO not
only serves as the source of carbon, it replaces H 2
and hydrogenase as the source of electrons for the
reductions (see Eqn. 1). Furthermore, the CO reacts directly with the Y site of CO-DH as illustrated by the cross-hatched arrow ( - x - x - ) of
Fig. 1. We will have more to say about the Y site
of C O - D H later.
co
.
ss..o
CH3
CO SCoA
@
t
=~'~
[ Anabolism
CH3COSCoA~
CO
l®
....... J
z
co,
H20/
@
CH3 [CC~ - CH3Tr
[Co']E
CO2
Acetate
...,
,r
,,,~/C H3THF
~-..,
CO2
6H++6e
3H2
Fig. 1. The acetyl-CoA pathway for autotrophic growth by acetogertic bacteria. THF is tetrahydrofolate, CH3Tr is methyltransferase,
CoE is corrinoid enzymes, ~
is CO dehydrogenase with 3 subsites, X, Y, Z. SS-Red is CO dehydrogenase disulfide reductase
and H2ase is hydrogenase. The broken arrow indicates anabolic reactions.
348
acetyl group is formed at the Y site via sequences
4, 5 and 6 of Fig. 1 and that in the absence of
CoASH and other enzymes, the acetyl group is
hydrolyzed to acetate. We propose that the CoASH
is added subsequent to this step. The enzyme, CO
dehydrogenase disulfide reductase, is required for
the addition of CoASH [26] (7 of the scheme). We
have suggested that an SSCoA linkage is formed
with CO-DH at the Z site which is followed by the
conversion to acetyl-CoA by the CO-DH (8 of the
scheme). It is evident from this scheme that
nickel-containing C O - D H is the central enzyme
of the acetyl-CoA pathway.
We know very little about the methyl group to
the C O - D H nor have we established that the
CoASH is bound to the enzyme in a disulfide
linkage. We have been able to treat the C O - D H
with 14CH3I and then remove the excess 14CH31
from the C O - D H . With this methylated C O - D H ,
CO and CoASH and dithiothreitol or CO disulfide reductase, [14C]acetyl-CoA is formed
without addition of CH3THF, methyltransferase
and the corrinoid enzyme [27]. This result offers
promise as a means of identifying the methyl site
on C O - D H .
Information has been obtained about the Y site
using electron spin resonance [21,28,29]. An ESR
signal is observed upon incubation of C O - D H
with CO which is from a spin-coupled center at
the Y site consisting of nickel, iron and carbon
derived from CO. This conclusion is based on
evidence obtained using 61Ni and ~3CO, which
demonstrate that the unpaired electron is associated with both atoms [28]. When 57Fe is substituted for 56Fe, there are strong hyperfine interactions [29] showing Fe also is at the Y site.
Furthermore, when the enzyme is treated with
both CO and CoASH or acetyl-CoA, there is a
substantial change in the signal [21]. It is concluded from these later studies that the Z site,
where the CoASH and acetyl-CoA bind, is close
to the Y site on C O - D H .
3.2. The acetyl-CoA pathway and other
bacteria
There are numerous aerobic and
bacteria that grow with CO z and H 2 as
of carbon and energy and several of
acetogenic
anaerobic
the source
these are
acetogenic. The aerobes use the Calvin cycle (reviewed by Bowien and Schlegel [30]). Among the
anaerobic bacteria, there are several acetogens
which quite certainly use the acetyl-CoA pathway
during growth. Some of these, as does C. thermoaceticum, apparently utilize the acetyl-CoA pathway during heterotrophic growth [10,31]. In addition, there are acetogenic bacteria which use
purines or glycine as a source of carbon and as a
reducing agent [32-34]. The latter do not use the
acetyl-CoA pathway; they use the glycine synthase
pathway [1,35-38] which may some day prove to
be used by autotrophs. These bacteria contain
high levels of tetrahydrofolate enzymes [36] but in
comparison to C. thermoaceticum and Acetobacterium woodii contain low levels of C O - D H and
corrinoids [39].
Among the acetogens, aside from C. thermoaceticum, A. woodii has been studied most extensively and we will review some of the results
obtained with this organism. A. woodii, when
grown on fructose, produces acetate in large quantities and when grown autotrophically, reduces
CO 2 in the presence of H 2 to acetate. It contains
high levels of C O - D H , corrinoids and enzymes of
the tetrahydrofolate pathway [39] and it has been
shown that cell-free extracts convert ~4CH3THF
or laCH3-B12 to [~4C]acetate during fermentation
of pyruvate [39]. The C O - D H [40] and phosphotransacetylase [41] have been purified, however, the other enzymes catalyzing the conversion
of CH3THF, CoASH and CO or CO 2 and H 2 to
acetyl-CoA have not been isolated from this
organism.
Much of the evidence for the acetyl-CoA pathway is indirect. It has been shown using [U14C]acetate and [2-14C]pyruvate as tracers with
cells growing with CO 2 and H 2 as the substrates
that they do not use the reductive pentose cycle or
a complete reductive tricarboxylic acid cycle [42].
Alanine, aspartate, and glutamate were isolated
from hydrolyzates of the protein and glucosamine
from hydrolyzates of the cell wall. The distribution of the ~4C in these compounds was not in
accord with predictions from the Calvin cycle but
were in accord with a utilization of the [U~4C]acetate by carboxylation via acetyl-CoA to
pyruvate followed by metabolism in an incomplete
349
tricarboxylic acid cycle. These authors also investigated the enzyme pattern of A. woodii [43].
Ribulose-l,5-bisphosphate carboxylase was not
present, again showing the bacteria do not use the
Calvin cycle. It was found that a-ketoglutarate
dehydrogenase/synthase was not present, thus
accounting for the incomplete Krebs cycle. The
results provided no direct information concerning
the mechanism of synthesis of acetate, but did
show that once synthesized, it could provide the
source material for growth of the organism.
A series of experiments were then done which
will be considered in relation to the the acetyl-CoA
pathway of Fig. 1. Diekert and Ritter [44] grew ,4.
woodii on CO 2 and H 2 and, during exponential
growth, added ~4CO to the gas phase. 9% of the
CO was converted to CO 2 and 89% of the CO was
present in the carboxyl position of acetate. It is
seen in Fig. 1 that CO enters via C O - D H and is
converted to the carbonyl group of acetyl-CoA.
CO 2, the precursor of the methyl group, was
unlabeled, thus the distribution of 14C was as
predicted from the scheme of Fig. 1.
An interesting study of the conversion of CO 2
and CO to acetate has been done using ~3C-nuclear
magnetic resonance (a3C-NMR) measurements
[45]. By this procedure, the relative amounts of
1 3 C H 3 - C O O H , C H 3 - 1 3 C O O H and 13CH313COOH were determined. When cells were grown
with ~3CO, and unlabeled CO 2 and with ~3CO2
and unlabeled 12CO, the results were in accord
with the prediction from the scheme in Fig. 1. It
was shown that CO is preferentially converted to
the carboxyl position and CO 2 to the methyl position of acetate. Similar results were obtained with
Butyribacteriurn methylotrophicum, which is an
acetogen [45].
Studies were done with cyanide since it is known
to inhibit the conversion of CO to CO 2 by C O - D H
[18,46]. Diekert et al. [47] demonstrated that when
A. woodii was grown with 14CO2 and H 2, CO was
produced, presumably by the C O - D H . When
cyanide (1 mM) was added, it inhibited formation
of both acetate and CO which is in accord with
the requirement that C O - D H is essential for formation of acetate from CO 2 (see Fig. 1). Experiments also were done with washed suspensions of
cells. Cyanide was found to inhibit the conversion
of ~4CO2 to acetate, formate and CO but it did not
inhibit the conversion of 14CO into acetate; if
anything, there was a slight stimulation. Since
cyanide did not inhibit the incorporation of CO,
the authors considered the only involvement of
C O - D H in the acetate pathway is to convert CO,
to CO. We think C O - D H has a far greater role.
We believe the cyanide inhibits the electron transfer required for the conversion of CO 2 to CO and
that there is no inhibition of the formation of the
C~ intermediate by C O - D H from CO or of the
other reactions which are proposed to be catalyzed
by C O - D H in the scheme of Fig. 1.
Clostridium therrnoautotrophicum is another acetogen which appears to use the acetyl-CoA pathway. It contains high levels of C O - D H , hydrogenase, corrinoids and tetrahydrofolate enzymes
when grown on H 2 and CO 2, methanol or glucose
[48]. Although extensive studies have not been
done, Clostridium formicoaceticum [10] and Clostridium aceticum [10] are acetogenic and most
likely use the acetyl-CoA pathway.
It is apparent that the overall results of these
studies with A. woodii are in accord with metabolism via the acetyl-CoA pathway, however, much
remains to be done to verify fully the role of this
pathway in A. woodii and in other acetogenic
bacteria.
4. T H E A C E T Y L - C o A
METHANOGENS
PATHWAY
AND
There are two types of methane bacteria, those
that oxidize and utilize methane for growth and
those that produce methane. The latter are called
methanogens. Methanogens can convert compounds such as CO, CO 2 and H 2, formate, methylamines, methanol and acetate to methane. We
will first consider the methanogens that grow
anaerobically with CO 2 and H E as the source of
carbon and energy and are autotrophs. Although
they are not acetogenic (i.e., produce acetate as a
major end product), the evidence is quite convincing that they use the acetyl-CoA pathway in
anabolism when grown on CO or CO 2 and H E.
We will also consider the methanogens that produce methane from methanol and from acetate.
350
Although this is not autotrophic growth, there is
evidence that growth on methanol or acetate does
involve the acetyl-CoA pathway. Methanogens are
archebacteria and they possess a battery of cofactors which differ from those of eubacteria. Some
replace functions of cofactors of the eubacteria
and others are specific cofactors involved in the
formation of methane (see [49] and [50] for recent
reviews).
We will not attempt to deal extensively with the
first type of evidence, it is reviewed by Zeikus [31].
Daniels and Zeikus [51] pulse-labeled cell suspensions of Methanobacteriurn thermoautotrophicum
with 14CO2. The ~4C was found in 1-carbon carriers and alanine, aspartate, and glutamate. Fuchs
and Stupperich [52] added [U-]4C]-succinate to
growing cells of M. thermoautotrophicum; they
disrupted the cells with a French press and isolated amino acids from the hydrolyzed protein.
The glutamate contained ~4C in carbons 2 to 5,
none in C-l, and the alanine and aspartate were
devoid of ~4C. The results indicate a-ketoglutarate
was synthesized by the reductive carboxylation of
succinyl-CoA but the conversion of a-ketoglutarate to isocitrate and cleavage of citrate to
oxalacetate and acetyi-CoA did not occur as would
be expected if metabolism involved a complete
reductive citric acid cycle. Overall evidence supporting these conclusions was obtained in similar
e x p e r i m e n t s using [U-]4C]acetate and [3~4CJpyruvate [53]. The results fit a mechanism
involving formation of pyruvate via fixation of
CO 2 with acetyl-CoA. These results and others
show that the reductive pentose cycle, the reducrive tricarboxylic acid cycle, the serine, and ribulose monophosphate pathways do not account for
the autotrophic growth of these methanogens.
4.1. The acetyl-CoA pathway and autotrophic growth
of rnethanogens with CO, and H,
There are two parts to the evidence that the
acetyl-CoA pathway is involved in autotrophic
growth on CO z and H 2 by methanogens. One is
indirect in that it has been shown that the autotrophic methanogens do not use the reductive
pentose cycle or the complete reductive tricarboxylic acid cycle. The second deals more directly
with the mechanism of acetyl-CoA synthesis. In
large part, both sets of evidence are based on
tracer studies with CO and CO 2 and inhibition
studies with KCN and alkyl halides. The key
enzymes of the acetyl-CoA pathway, except for
C O - D H and the corrinoid protein, have not been
isolated from methanogens and no studies have
been done with the isolated enzymes to show they
are involved in acetyl-CoA synthesis.
CO 2
~.~FormyI-~.~_~._.~CHI-H4MPT~
#
,6e
,"
~'~ Alkyl halides
/
[ColE
" " "' - ~ 4H2
CH3SCoM
=
CH4
J
'~
CH3[Co]E
H2"
/
".~.
co2,.,2H*,2~
'
. .'
"..
CN
co
X
)'
">--"
Z
'
CoASH ...................
CH3
co
"
-.
",
CH,jCO SCoA
"lh"-- " 4 ~ ~ ~ " ~ ' ~
~ a'- C H 3 C O s C O A
H#)
',
'..
,'
CO
""
t
["Anab°liSm 1
Fig. 2. Outline of autotrophic pathway for growth with CO 2 and H 2 or CO by methanogens, MFR is methanofuran, H4MPT is
tetrahydromethanopterin, CoM is 1 mercaptoethanosulfonic acid, CN is cyanide and other abbreviations are given in the legend to
Fig. 1. Broken arrows indicate anabolic reactions.
351
We will now turn to the more direct evidence
that growth by methanogens occurs with formation of acetyl-CoA from 2 molecules of CO 2 which
is then used as the starting material for anabolism.
in Fig. 2, we have expanded schemes proposed by
Stupperich and Fuchs [54] Ri3hlemann et al. [55]
and Evans et al. [56] to indicate the extensive role
that CO-DH may have in the pathway. It is proposed (i) that C O - D H serves as the CO, CH3,
SCoA acceptor and catalyzes the final steps of the
synthesis of acetyl-CoA: (ii) that the corrinoid
enzyme ([Co]E) serves as a methyl carrier between
the pathway for synthesis of methane (top of Fig.
2) and that of acetyl-CoA synthesis (bottom of
Fig. 2); (iii) that H 2 is the electron donor since
growth is on CO 2 and H2; (iv) that methyltetrahydromethanopterin (H4MPT) is the methyl donor
to [Co]E. This latter suggestion is in accord with
the recent demonstration by Lange and Fuchs [57]
showing that methenyl-H4MPT is converted to
the methyl of acetyl-CoA by an extract of M.
thermoautotrophicum.
It is beyond the scope of this re~4ew, which is
primarily concerned with the acetyl-CoA pathway,
to attempt to deal comprehensively with the mechanism of formation of methane. Wolfe [50] has
recently reviewed the cofactors involved in the
conversion of CO 2 to methane. The first stable
product has been identified as formylmethanofuran (MFR of Fig. 2) [58], the formyl group is
then reduced and converted via several steps to
methyltetrahydromethanopterin (CH3H4MPT of
Fig. 2) [59]. Tetrahydromethanopterin has a complex structure containing a pterin ring which is
similar to that of folate. The methyl is then transferred to 2-mercaptoethanosulfonic acid (HSCoM
of Fig. 2) [60]. The last step is the reduction to
methane which in itself involves four protein components (only one of which has been purified):
Mg 2~, ATP, FAD, F420 (a deazaflavin derivative)
[611, F430 (a nickel tetrapyrrole) [62] and an unidentified component B. Clearly, this portion of
the scheme of Fig. 2 is complex and unique. For
the formation of acetyl-CoA during growth on
CO2 and H2, we propose that some of the methyl
H 4 M P T is used to form the C-2 of acetyl-CoA. In
this sequence, we propose that the methyl of
methyl-H4MPT is transferred to a corrinoid pro-
tein. From here, the reactions are the same as for
the acetogens. The methyl is transferred to
C O - D H and C O - D H condenses the bound CH~,
CO and SCoA groups to form acetyl-CoA.
It should be noted that, if the overall mechanism is as shown in Fig. 2, the formation of
methane (the top portion of the scheme) can occur
independently of the formation of acetyl-CoA.
The acetyl-CoA portion of the pathway supplies
the acetyl-CoA for the anabolic reactions (shown
by dashed arrows). The methyl group is derived
from the methane portion of the mechanism. It is
considered that the acetogens obtain the necessary
energy for growth with CO 2 and H 2 by formation
of acetate: whereas, the methanogens derive their
energy by forming methane [51].
The question we will now address is how well
does the scheme of Fig. 2 meet the requirements
of presently available information on acetyl-CoA
synthesis by methanogens using CO 2 and H 2. We
have selected for consideration some of the more
recent investigations of this pathway.
Convincing evidence has been presented by
Ri~hlemann et al. [55] that acetyl-CoA has a pivotal
role in CO 2 assimilation. They pulse-labeled growing cells of M. thermoautotrophicum with 14CO2
and identified the resulting [~4C]acetyl-CoA by
several methods, including measurement of its activity in the citrate synthase reaction and by using
HPLC to compare acetyl-CoA and CoA with
authentic acetyl-CoA and CoA. The kinetics of
the labeling of the acetyl-CoA from ~4CO2 showed
that acetyl-CoA is an initial product of CO 2 fixation. The amount of acetyl-CoA was small, 0.1
n m o l / m g dry weight of cells. These findings are
extremely important since Leigh [63] had found
little or no pantothenic acid in methanogens which
cast doubt on the central role of acetyl-CoA in
their metabolism.
Stupperich and Fuchs [54,64] studied the
synthesis of acetyl-CoA using an in vitro system at
60°C under 80% H 2 and 20% ~4COz containing a
cell-free extract of M. thermoautotrophicum, 1,4piperazinediethanesulfonic acid (Pipes) buffer, pH
6.7, MgCI 2, ATP, CH3SCoM, CoA, dithiothreitol
and ferrous ammonium sulfate. They report [64]
that omission of ATP reduced the yield of acetylCoA and of methane each about 50%; whereas,
352
omission of CoA reduced the yield of acetyl-CoA
about 66% but had no effect on the yield of
methane. If CH3SCoM was omitted almost no
acetyl-CoA or methane was formed. HSCOM was
ineffective. Bromoethanesulfonic acid, an analogue of CoM, inhibited formation of both acetylCoA and methane almost completely.
When extracts were treated with H 2, 14CO2
and CO in the presence of cyanide, acetate was
formed and the 14C was converted to the methyl
of acetyl-CoA but none to the carboxyl position
[54]. These observations are in accord with the
scheme of Fig. 2 and we assumc, as proposed by
Stupperich and Fuchs [54], that cyanide inhibits
the reduction of CO, to CO (1 of Fig. 2) and
therefore 14('O, cannot be converted to the
carbonyl group of acetyl-CoA. On the other hand,
the CO can combine with the C O - D H even though
cyanide is present (2 of Fig. 2) and the remaining
reactions of the C O - D H are not inhibited by
cyanide. Thus, [2-14C]acetyl-CoA can be synthesized since CO is converted to the carbonyl group
and 14CO2 can be converted to the methyl group
via the top sequences of Fig. 2. CO~ also can be
converted to methane since the C O - D H is not
directly involved in methane synthesis. If CO is
omitted in the presence of cyanide, acetyl-CoA
synthesis can no longer occur since there is no
source for formation of the carbonyl group. Thus,
under these conditions, CO 2 is not converted to
the methyl of acetyl-CoA but methane formation
is uninhibited [54].
Even though CH3SCoM was necessary for
acetyl-CoA synthesis, the 14C of 14CH3SCoM was
not converted to acetyl-CoA. However, 40% of the
methane formed was from 14CH3SCoM and 60%
was from unlabeled CH3SCoM formed from CO 2
[54]. These results show, as indicated in Fig. 2,
that the requirement for CH3SCoM is not for the
direct synthesis of the methyl of acetyl-CoA. The
indirect requirement of CH 3SCoA for synthesis of
acetyI-CoA may be related to the so-called R P G
effect. Wolfe and his collaborators have shown
that the rate of production of methane from CO~
is increased 30-fold over that by extracts not supplemented with CH3SCoM and have called it the
R P G effect [65]. The explanation of this effect
remains unknown [49].
In vivo tests were also done with cells growing
with CO_, and H= [54]. When the optical density
of the cells was about 1, if 0.2 mM KCN was
added, growth ceased but methane production
continued. When CO was included in the gas
phase, growth continued and increased with increasing concentration of CO. When the gas phase
was 20% CO, there was very little inhibition of
growth by cyanide. When 14CO was used, 14C was
incorporated by the cells. The alanine from the
cells was degraded and C-2 of the alanine contained 74% of the 14C of the molecule and with a
specific activity of 81% of that of the 14CO. These
results show that the CO can bypass the cyanide
inhibition of the C O - D H just as was observed in
the in vitro experiments and acetyl-CoA synthesis
and growth was thus possible. The CO was almost
certainly converted by the cells to the C-1 of
acetyl-CoA then to pyruvate and then to alanine;
thus, the in vivo results are in accord with the in
vitro studies.
Inhibition studies were done with alkylhalides.
Alkylhalides are known to inhibit corrinoid
enzymes and the inhibition is removed by exposure to light [66]. Holder et al. [67] observed in
an in vitro system similar to that described above,
that 10--20 p.M propyl iodide strongly inhibited
acetyl-CoA formation from CO~ and H I but had
little effect on formation of methane. In the presence of light, there was no inhibition of acetate
formation. With growing cells, 1 p.M and 2 p.M
propyl iodide had little effect on growth but there
was increasing inhibition at 5 and 10 p,M and.
with 40 p,M, there actually was a decrease in cell
density. However, methane formation was inhibited only slightly even by 40 p.M propyl iodide. In
the presence of light, 40 p.M propyl iodide had
little effect on growth.
The above results with extracts and with growing cells are in accord with the scheme of Fig. 2.
The inhibition of the corrinoid enzyme by the
propyl iodide inhibits transfer of the methyl from
the C H 3 - H 4 M P T to [Co]E (3 of Fig. 2) and thus
formation of acetate is inhibited. In light when the
inhibition of the corrinoid by the propyl iodide is
prevented, the formation of acetyl-CoA is no
longer inhibited. Since methane formation (as
shown at the top of the scheme) does not directly
353
involve the corrinoid enzyme, alkylhalides would
be predicted not to inhibit the formation of
methane.
The results with methyl iodide were quite different. With extracts, acetyl-CoA formation actually increased in the presence of methyl iodide (50
/xM), being about double that formed in the control without methyl iodide. Methane formation
was unaffected by methyl iodide. It appears the
methyl iodide may have combined with the cobalt
of the corrinoid enzyme and thus served as a
substrate for the formation of the methyl group of
acetyl-CoA.
With whole cells, methyl iodide, like propyl
iodide, inhibited growth but did not inhibit
methane formation and the inhibition of growth
was eliminated by light. Since CH3I did not inhibit formation of acetate by extracts, it was concluded that the reversible inhibition of growth by
CH~I could not have been due to inhibition of a
corrinoid enzyme involved in the formation of the
acetyl-CoA. Thus, the inhibition of growth by
CH~I was postulated to be via a second corrinoid
which is required for growth [67]. The proposal
that there may be a second corrinoid (or metal
center) involved in some reaction required for
growth does not alter the fact that most, if not all,
the evidence is in accord with the proposed acetylCoA pathway.
Recently, an interesting procedure for study of
the metabolism of acetate by M. thermoautotrophicum was reported in which 13C-NMR of 2,3
cyclopyrophosphoglycerate (CPP) was used as a
monitor. It had been shown by 13C-NMR that
[I-13C]acetate is incorporated specifically into C-2
of CPP, [2-13C]acetate in C-3 and [1-13C]pyruvate
into C-1 [68]. In the most recent study [56] 13CN M R was used to determine scrambling of the
carbons of [1,2-13C]acetate and [2,3-13C]pyruvate
that occurs when the compounds are incorporated
into CPP during growth of M. thermoautotrophicure. Scrambling indicates the acetate or pyruvate
had been degraded to C~ units which exchanged
with the lZCO2, thus resulting in the introduction
of a ~2C next to a ~3C during resynthesis and
conversion to CPP. It was found that scrambling
of the aSC-~3C does occur and that ~2C is introduced into C-2 of CPP [56]. The effect of cyanide
and propyl iodide on the scrambling was determined. It was found with cyanide present, [1,2t3C]acetate was incorporated into CPP without
scrambling but. in the presence of propyl iodide,
scrambling occurred. These results are in accord
with the predictions from Fig. 2, since cyanide
inhibits the conversion of CO to CO 2 by C O - D H .
Thus, ~2CO~ could not be converted to 12CO and
replace the 13C of the carbonyl group of acetylCoA by the reversible exchange of CO with acetylCoA as catalyzed by CO DH [21] (see 2 of Fig. 2,
and Eqn. 2). Propyl iodide does not inhibit the
scrambling since the corrinoid enzyme is not
involved in the conversion of CO, to CO by the
C O - D H or the exchange of the resulting CO with
acetyl-CoA. These observations are in accord with
the view that C O - D H per se of the methane
bacteria may catalyze the final step of the formation of acetyl-CoA and an exchange reaction as
has been observed with C. thermoaceticum.
In summary, most observations that have been
made with CO 2 and H 2 as substrates for methane
bacteria, appear to be in accord with the synthesis
of acetyl-CoA as illustrated in Fig. 2.
4.2. The acetyl-CoA pathway and growth of
methanogens with methanol
Certain methanogens can grow with methanol
as the substrate and the stoichiometry of the conversion is as follows:
4CH3OH ---, CO 2 + 3CH 4 + 2 H 2 0
(3)
We will see that there are conflicting data concerning the metabolism of methanol, particularly
with regard to the role of corrinoids in the formation of methane. In addition, the mechanism of
the oxidation of methanol to CO 2 apparently has
not been determined with certainty. It has been
proposed that the methanol may be converted to
CO 2 by reverse of the reactions which occur when
CO 2 is reduced to a methyl group although it is
considered possible that the methanol may be
oxidized directly to CO 2 [31,49]. Anabolism is
considered to occur by formation of acetyl-CoA as
a precursor of cell carbon. For purposes of discussion, a scheme is presented in which it is assumed
that 4 molecules of methanol are converted by a
methyltransferase to the methyl corrinoid enzyme
354
r'"
~
sMFR
~
FO r mY' "~"~" ~
. . ~ " H4 M P T --..~
T ~ - - . ~ =
\
~1~1 hol.de~.
/....f---- H SCo M " ~
6H + , 6e
/
CO 2
2H +. 2e
,
. , . . _ _ . . ~ CN
4..~
H20
/
I
co
.~ CoASH --.- . . . . . . . . .
\
CH3 CO
\
....
X y Z
-~..- ~
:
CH3 CO SCoA
"-
X y
t~....,_~ ~
Z
"--'~.-""
CH3COSCo A
CO
Fig. 3. Outline of pathway of growth of methanogens using unethanol as the substrate. Abbreviations are as in the legends of Figs. 1
and 2. Broken arrows indicate anabolic reactions.
as indicated in Fig. 3. Then. 3 molecules of the
CH.~[Co]E are converted to the CH3SCoM and
then to methane and one of the 4 molecules of
C H 3 [Co] E is converted via H 4 M P T and M F R to
CO 2. These conversions constitute the catabolic
reactions. The anabolic reactions shown with
broken arrows (Fig. 3) involve the synthesis of
acetyl-CoA via C O - D H using CO 2 and the methyl
from the CH3[Co]E that are generated in the
catabolic reactions. It is to be noted that the
overall stoichiometry shown in Eqn. 3 does not
take into account the methanol that is used for
anabolism. However, the amount used for anabolism is small compared to the total that is
metabolized during growth.
The scheme of Fig. 3 will now be considered in
relation to observations that have been reported
concerning methanol metabolism. There is considerable evidence that the methanol is converted
to a methyl on a corrinoid prior to conversion to
methane. Blaylock and Stadtman [69] reported
that methylcobalamin is formed from Cob(I)alamin and methanol and the system later was resolved from Methanosarcina barkeri into four
components [70]. Taylor and Wolfe [71] have
purified a methyltransferase from Methanobacteriurn bryantii that catalyzes the transfer of the methyl
group from methyl-B12 to HSCoM. Wood et al.
[72] purified a methyl B~2-containing protein from
M. barkeri grown on methanol but did not assay
to determine if it had transferase activity. Van der
Meijden et al. [73] have purified a corrinoid pro-
tein which catalyzes the methylation of its corrinoid with methanol. The enzyme is designated
methanol: 5-hydroxybenzimidazolylcobamide
methyltransferase (abbreviated MTt). They also
have purified an enzyme from M. barkeri that
catalyzes the transfer of the methyl group of CH 3B~2 or from the methyl group of methylated MT~
to HSCoM [74,75] and have named this enzyme
Co-methyl-5-hydroxybenzimidazolylcobamide :
HSCoM methyltransferase (abbreviated MT 2). The
methyl of the CH3SCoM is then converted to
methane by the methyl reductase system. The
cobalt of MT 1 must be in the reduced Co 1÷ state
for the enzyme to be active. This is accomplished
with a reducing system consisting of H 2, ferredoxin. F420 and hydrogenase. In the formation of
acetyl-CoA from methanol, we propose that the
methyl of the methyl corrinoid enzyme is transferred to C O - D H . The C~ is formed by reduction
of CO 2. Then C O - D H combines CoA with the
bound C~ and methyl group to form acetyl-CoA.
It would be expected if the mechanism occurs
as shown in Fig. 3, that alkylhalides would inhibit
the formation of CH3-MT 1 ([Co] E of Fig. 3) and,
thereby, the formation of methane and acetyl-CoA.
Eikmanns and Thauer [76], however, found that
propyl iodide did not inhibit methane formation
from methanol with cell suspensions of M. barkeri
but did inhibit the exchange of CO 2 with acetate.
Kenealy and Zeikus [77], found with cell suspensions of M. barkeri, that propyl iodide did not
inhibit the synthesis of CH3SCoM but inhibited
355
the synthesis of acetate from 14CO. They did not
report the effect on methane formation. These
inhibitions by propyl iodide were prevented by
exposure to light which is considered evidence the
inhibition is caused by inactivation of the corrinoid.
Shapiro [78] found that both the conversion of
the methyl of methanol and of CH3B~2 to
CH3SCoM were not inhibited by propyl iodide.
However, the alkylhalides did inhibit the conversion of methanol to methane. He proposes
corrinoids are not involved in the formation of
CH3SCoM from methanol and that alkylhalides
inhibit the reduction of C H 3 S C o M to methane.
Possibly, this inhibition of methane formation
could result from the reaction of the alkylhalide
with the nickel of the tetrapyrrole of F43o. We
have found no reports, however, indicating that
1=43o reacts with alkyi halides. Recently, Whitman
and Wolfe [79] have reported that corrins activate
the methylreductase system from M. bryantii
three- to five-fold in extracts resolved from low
molecular weight factors.
Clearly, it is difficult to reconcile all the observations that have been reported on the conversion of methanol with the scheme of Fig. 3. On
the one hand, the recent studies with purified
enzymes by Vogels and co-workers and previous
studies with enzymes indicate corrinoids are directly involved in the formation of methane from
methanol in accord with the scheme shown in Fig.
3. On the other hand, inhibition studies with alkylhalides, indicate the corrinoids are not involved
in methane formation but are involved in the
synthesis of acetate, since acetate formation (CO z
exchange) was inhibited. Clearly, no decision is
possible at this time concerning the route of
methanol conversion to the methyl group of
acetate.
We wish to point out, however, that inhibition
by alkylhalides may not be a completely reliable
indication of whether or not a corrinoid is involved.
For example, Thauer et al. [80] and Diekert and
Thauer [46], based on results of alkylation and
photoreactivation, considered that clostridial
C O - D H was a corrinoid enzyme. Subsequent
studies have shown C O - D H is a nickel enzyme
and it is not a corrinoid enzyme [18]. Thus, it is
clear that studies by alkylation and photoreactivation must be interpreted with caution. The studies
by Vogels and co-workers with purified enzymes
arc quite convincing that there is methylation of a
corrinoid and it is involved in methanogenesis
from methanol as indicated in Fig. 3.
4.3. Catabolism of aceo'l-CoA by methanogens
We will now consider the metabolism of
methanogenic bacteria which use acetate as a
source of carbon, M. barkeri has been the most
thoroughly investigated. The products of acetate
catabolism are CO 2 and methane. These organisms
also grow autotrophically with CO 2 and H 2
[16,31,49,81,82].
Our concept of the pathway for formation of
methane and acetyl-CoA from acetate is presented
in Fig. 4. It is proposed that the acetate is converted to acetyl-CoA which is used for anabolic
reactions and for formation of methane by combination with C O - D H at the X, Y. Z sites (1 of Fig.
4). Then, the SCoA group is removed from the Z
site by a disulfide reductase (2 of Fig. 4), the C H 3
group is transferred to the corrinoid enzyme (3 of
Fig. 4) and the CO of the Y site is oxidized to CO2
by the C O - D H (4 of Fig. 4). Through the action of
a methyltransferase, the methyl group is transferred from a corrinoid protein to H 4 M P T (5 of
Fig. 4) which in turn reacts with HSCoM forming
CH3SCoM (6 of Fig. 4). It is proposed the
CH3SCoM is reduced by the methyl reductase
system to methane using electrons generated by
C O - D H during the conversion of Y-CO to CO2 (7
of Fig. 4).
There is a very significant difference between
the pathway for growth on H 2 and CO 2 and for
growth on acetate. Methanogenesis from H2 and
CO 2 (see Fig, 2) does not directly involve the
acetyl-CoA pathway The formation of methane is
the main metabolic pathway by which ATP is
generated for use in the synthetic reactions of
anabolism. The methyl group for the acetyl-CoA
pathway is supplied by the methane portion of the
sequence and the acetyl-CoA pathway serves to
provide the acetyl-CoA from which the anabolic
reactions are initiated. However, with acetate as
the substrate (Fig. 4), the acetyi-CoA pathway is
directly involved in the formation of methane and
356
C H3COOH "-~
-CH 3CO SCoA
CoASH, A T P - ~
H20, ADP ..,_i l
CH3COSCoA
t
r; n.bo".m I
Q
,
.
i
i,~"~"~
CoA SH
)
--'-'~<~
J
CO 2
i
2H +
x
co
,
H20
y@
C..H4 ~
i,
- - - - - ~ ' ~
,<-,
I
CHACO
@
\
Alkyl
hohdes
/ CH,~[Co ] E
CH 3 -H4MPT . . . .
SCoM
CH3"SCoM
./-
Fig. 4. Outline of pathway for growth of M. barkert using acetate as the substrate. Abbreviations are as in the legends of Figs. 1 and
2. Broken arrows indicate anabolic reactions.
the acetyl-CoA pathway becomes the major
metabolic pathway. In this pathway, C O - D H is
the central enzyme of the catabolic pathway. This
is in accord with the finding of Krzycki et al. [83]
that the activity of C O - D H is 5 × higher in cells
grown on acetate than in cells grown on CO~ and
H : or methanol. It has been proposed that ATP is
generated by electron and proton phosphorylation
[84]. Very recently, lvey and Ljungdahl [85] have
purified the F I portion of the H+-ATPase from C.
therrnoaceticum and, in whole cells, the FtF 0ATPase catalyzed the synthesis of ATP in response to a pH gradient.
Thus far, the only enzyme of the acetyl-CoA
pathway that has been isolated from M. barkeri is
C O - D H [80]. However, Krzycki et al. [84,86] have
obtained a 'soluble' enzyme system from M.
barkeri that converts acetate to CO 2 and methane.
The reaction mixture consists of the cell extract,
ATP, MgC12 and acetate with a gas phase of H 2.
They showed that methane and CO 2 originate
primarily from the methyl and carboxyl groups of
the acetate, respectively [86]. They demonstrated
[87] that acetyl phosphate replaces the requirement for both acetate and ATP. With [2i4C]acetate, H 2 and ATP, 14CHaSCoM was identified as a product. Bromoethanesulfonic acid, an
inhibitor that blocks utilization of CH3SCoM,
greatly reduced the rate of formation of methane
as did cyanide which inhibits C O - D H . The addition to the extract of an antibody to C O - D H
inhibited the formation of methane and C O - D H
activity. These results are all in accord with the
scheme of Fig. 4 and provide strong evidence for a
role of the acetyl-CoA pathway in acetate
metabolism.
Hydrogen was required in this soluble system
whereas it is not required with a particulate preparation described by Baresi [87]. It has been
suggested [84,86] that disruption of the membrane
bound electron transport system may lead to the
requirement of H2 in the soluble system.
We will now consider studies done with cell
suspensions of M. barkeri [76,88] and the effect of
cyanide and of propyl iodide on the reactions.
Eikmanns and Thauer [88] report that KCN (40
p,M) inhibited formation of methane from acetate
but not from CO 2 and H 2. These results are in
accord with the scheme of Fig. 2 in which C O - D H
has no direct part in the formation of methane
from CO 2 and H 2 and with that of Fig. 4 in which
C O - D H does have a direct role in the formation
of methane from acetate. The conversion of Y - C O
to CO 2 by C O - D H (4 of Fig. 4) is required for the
357
reduction of the CH2SCoM to methane and
cyanide inhibits this essential step.
These investigators also observed that cyanide
inhibited the exchange of CO 2 into acetate. We
have shown that C O - D H per se catalyzes an
exchange of CO with acetyl-CoA [21]. The same
type of exchange can occur with acetyI-CoA and
14CO, but in this case the CO 2 must be reduced
by the C O - D H to the CO level before it can be
converted to the carbonyl of acetyl-CoA. Apparently, it is the inhibition by cyanide of the
electron transfer involved in the conversion of
CO2 to CO that prevents the exchange of t4CO2
with acetyl-CoA.
Eikmanns and Thauer [76] have reported that 5
~M propyl iodide inhibited the formation of
methane from acetate by cell suspensions of M.
barkeri and that the inhibition was abolished by
light. These results indicate a role for corrinoids in
the formation of methane from acetate. It is apparent from the scheme of Fig. 4 that the inhibition of the corrinoid enzyme by propyl iodide
would prevent transfer of the methyl to the corrinoid enzyme (3 of Fig. 4) and thus inhibit the
formation of methane from acetate. Clearly. propyl iodide would have no effect on the exchange
of CO, with the carboxyl of acetate since that
exchange is catalyzed by C O - D H as explained
above (see 1 of Fig. 4. and Eqn. 2).
Eikmanns and Thauer [88] have investigated
the effect of CO on the conversion of acetate to
CO 2 and methane by cell suspension of M. barkeri.
The explanation of the results is not straightforward. They report that a 1% concentration of CO
in the gas phase completely inhibited methane
formation from acetate and that the rate of exchange of ~4CO2 with acetate was inhibited 50%..
The exchange of CO with CO 2 involves only COD H whereas the conversion to methane requires
the complete set of reactions of Fig. 4. Possibly,
CO may have more than one site of inhibition and
thus partially inhibit the CO exchange but completely inhibit formation of methane. We have
proposed that CO (4 of Fig. 1) and the acetyl
group of acetyl-CoA (8 of Fig. 1) both bind at the
Y site of CO-DH. Thus. CO may complete with
the binding of acetyl-CoA at the Y site and partially inhibit the exchange of CO with acetyl-CoA.
We have no suggestions concerning the second
possible site of inhibition that might be the cause
of the complete inhibition of methane formation.
In conclusion, with the possible exception of
the effects of CO on the system, where our information is not sufficient for a clear assessment, the
results so far obtained are in accord with the
proposed scheme of Fig. 4 for the conversion of
acetate to methane and CO~. The completely soluble enzyme system of Krzycki and Zeikus [84]
from M. barkeri, that converts acetate to CO~ and
methane, should provide an opportunity for isolation of enzymes and delineation of their role in
the pathway.
5. T H E A C E T Y L - C o A P A T H W A Y A N D T H E
S U L F A T E - R E D U C I N G BACTERIA
Recently, it has been clearly established that
certain sulfate reducing bacteria can grow autotrophically. Widdel [89] and Widdel et al. [90]
have isolated a pure culture of a sulfate-reducing
bacterium, Desulfonema limicola, which grows with
CO, and H 2 as the source of carbon and energy
and Klemps et al. [91] have shown that Desulfotornaculum orientis can grow autotrophically.
Jansen et al. [92] have conducted the only study
we are aware of to determine whether or not the
sulfate reducers use the acetyl-CoA pathway. The
organism which they used, Desulfoeibrio baarsii, is
by our definition, not a strict autotroph, since it
requires formate for growth and cannot use H 2 as
the electron donor. Formate plus sulfate serve as
the energy source and formate and CO 2 as the
source of carbon.
The medium which Jansen et al. [92] used contained inorganic salts, formate, sulfate, bicarbonate and the gas phase was 80% N 2 and 20% CO2.
[U-14C]acetate, [14C]formate, 14CO2 and 14CO
were used as tracers. During the growth with
[lac]formate the culture was gassed with N 2 / C O 2
to remove 14CO~ which was formed due to a rapid
cxchange of CO~ with formate. Alanine, aspartate
and glutamate were isolated from the hydrolyzed
protein of the cells and glucosamine from the
cell-wall fraction. With [U-~4C] acetate, the glucosamine had about twice the specific activity as the
358
alanine. It, therefore, is suggested that the synthesis of the glucose was via the E m b d e n - M e y e r h o f
pathway following conversion of the acetyl-CoA
to pyruvate by CO, fixation. In addition, ribulose1,5-diphosphate carboxylase was not found and it,
therefore, is concluded that the assimilation of
CO 2 did not occur via the reductive pentose cycle.
The distribution of ~4C in the alanine with
[U-~4C]acetate was 4% in C-l, 41~ in C-2 and
48% in C-3. This result is in accord with the
addition of CO 2 to [U-~4C]acetyl-CoA to form
pyruvate from which the alanine was synthesized.
When the source of ~4C was formate, the distribution of 14C in the alanine was 5% in C-l, 28% in
C-2 and 67% in C-3. The preferential labeling of
C-3 is that expected if acetyl-CoA was synthesized
by the acetyl-CoA pathway of Fig. 1 and then the
acetyl-CoA was converted to pyruvate by fixation
of CO:.
When t4CO was used with CO, and formate,
the distribution of the ~4C in the alanine was 15%
in C-l, 72% in C-2 and 14% in C-3. This distribution is in accord with CO entering the Y site of
C O - D H and then being converted to C-1 of
acetyl-CoA with formate being the source of the
methyl groups as in Fig. 1 followed by conversion
of the acetyl-CoA to pyruvate. The distribution of
~4C in aspartate was 9% in C-I, 46% in C-2, 35%
in C-3 and 10% in (7-4. This distribution is that
predicted if the pyruvate was carboxylated to form
oxalacetate in which the a4C was in C-2 of the
oxalacetate, then was partially randomized by
equilibration with symmetrical C4 dicarboxylic
acids prior to conversion to aspartate.
In conclusion, D. baarsii contains a very active
C O - D H and the authors conclude it synthesizes
its cell carbon from C~ compounds via an
'activated acetic acid pathway'. Further studies
are needed to more firmly establish the acetyl-CoA
pathway and investigations are needed to demonstrate the occurrence of the pathway in D. limicola
and D. orientis which grow on CO 2 and H2.
6. C O N C L U D I N G R E M A R K S
We have described in this review the acetyl-CoA
pathway of assimilation of CO 2 and its role in a
variety of organisms. This pathway not only has a
role in autotrophic growth from CO 2, it is an
important heterotrophic pathway by which
bacteria degrade organic material and convert it to
methane. The acetyl-CoA pathway thereby has an
important role in the carbon cycle. We are not
aware of any studies which indicate how much
carbon is cycled through the acetogenic bacteria.
Such studies would be of great importance in our
understanding of the role of the acetyl-CoA pathway in ecology. It is reported that, on a molar
basis, about 5% of the carbon fixed by photosynthesis is converted to atmospheric methane
[93]. The methanogens utilize the acetate that is
formed by other bacteria from sediments in
swamps, in the oceans and from organic layers in
the forest beneath the surface. One half of the
acetate is converted to CO 2 and the other half to
methane. Much of the methane that is produced is
reoxidized by aerobic forms at the surface to CO 2.
Methanogens, thus, have a very significant role in
the total carbon cycle.
Unlike the reductive pentose and reverse tricarboxylic acid cycles of assimilation of CO 2. the
acetyl-CoA pathway is not a cycle; it occurs by
direct conversion of two molecules of C(), to
acetyl-CoA, one of which is reduced to the methyl
group. The master enzyme of the pathway is
carbon monoxide dehydrogenase. It converts one
CO 2 to the CO group, is the acceptor of the
methyl and CoA groups and converts them to the
acetyl-CoA. We suggest that this enzyme be called
acetyI-CoA synthase to differentiate it from the
carbon monoxide dehydrogenases of aerobic bacteria and photosynthetic bacteria which use the
Calvin cycle. The roles of the two enzymes are
very different. The enzyme of the aerobes and the
photosynthetic bacteria serve to convert CO to
CO 2 and that of the anaerobes to catalyze the
synthesis of acetyl-CoA. The C O - D H of Rhodospirillum rubrum, which is a nickel enzyme, does not
catalyze the exchange reaction between CO and
the carboxyl of acetyl-CoA (S.W. Ragsdale, D.
Bonam and P. Ludden, unpublished results).
The assimilation of CO 2 by the acetyl-CoA
pathway involves a remarkable number of metallo-enzymes. The formic dehydrogenase contains
selenium and tungsten or molybdenum and iron
359
sulfur centers, the hydrogenase is an iron-sulfur
enzyme, the corrinoid enzyme is a cobalt,
iron--sulfur-containing enzyme and the CO dehydrogenase is a nickel, zinc, iron-sulfur enzyme.
The exact role of these metals in the catalysis of
the acetyl-CoA pathway is under investigation.
We are not experts in methanogenesis, and
trust that the saying, 'Fools rush in where angels
fear to tread' does not apply to our efforts. An
important question to be answered is whether
C O - D H functions in methanogens as in C. thermoacticum in catalysis of the final steps of the
synthesis of acetyl-CoA. The C O - D H of methane
bacteria, that has been purified, has a subunit
structure which differs from the C O - D H of C
thermoaceticum. It has a M~ of 232000 and is
made up of two different subunits of M~ 18000
and 92000 [81] whereas the C O - D H of C. thermoaceticum [18] and A. woodii [40] have almost
identical M~, and subunits of approx. 80000 and
70000. The ( ' O - D H of C. thermoaceticum, as
isolated, includes all the sites necessary to catalyze
the final combination of the methyl, carbonyl, and
CoA to form acetyl-CoA. In contrast, the C O - D H
of the methane bacteria, as isolated, may catalyze
only a portion of the final steps. In this regard, the
observations of Bott et al. [94] are of interest.
They report that Methanobret'ibacter ruminantum.
Methanobrevibacter hinithii and Methanococcus
eoltae, which are heterotrophs, do not contain
C O - D H , in contrast to autotrophic forms. However, Methanospirillum hungatei which does conrain C O - D H was unable to grow on CO 2 and H 2
and required acetate for growth. This may indicate
that the C O - D H of some methane bacteria lack
of portion required for the overall process. The
situation may be similar to that of biotin enzymes.
Pyruvate carboxylase contains all the catalytic sites
required for the final synthesis on each peptide
chain. However, transcarboxylase and acetyl-CoA
carboxylase have the catalytic sites on separate
subunits of different primary structure which, in
combination, catalyze the overall reaction [95].
Additional studies will be required to ascertain
whether the C O - D H of the methane bacteria
requires some additional component to catalyze
the overall synthesis of acetyl-CoA. It is our prediction, however, that the C O - D H of methanogens
will be found to be involved in some part of the
final steps of the synthesis.
ACKNOWLEDGEMENTS
Work in our laboratories on the acetyl-CoA
pathway is supported by Grant G M 24973 from
the National Institutes of Health.
REFERENCES
[1] Wood, H.(.;., Ragsdale, S.W., and Pezacka. E. (1986) The
acetyl-CoA pathway: a newly discovered pathway of autotrophic growth. Trends Biochem. Sci. 11, 14-18.
[2] Schlegel, H.G. (1975) Mechanism of chemo-autotrophy, in
Marine Ecology (Kline, O. Ed.) Wiley, New York, Volume
2, pp. 9-60.
[3] Quayle, J.R. and Ferenci, T. (1978) Evolutionary aspects
of autotrophy. Microbiol. Rev. 42, 251-273.
[4] Colby, J., Dalton. H. and Whittenbury, R. (1979) Biological and biochemical aspects of microbial growth on (71
compounds. Ann. Rev. Microbiol. 33, 481-517.
[5] Whittenbury, R. and Kelly, D.P. (1977) Autotrophy: a
conceptual phoenix, in Microbial Energetics Symposium
27. Soc. Gen. Microbiol. (Haddock, B.A. and Hamilton,
W.A., Eds.) pp. 121-150. Cambridge University Press,
Cambridge.
[6] Buchanan, B.B. and Sirevag, R. (1976) Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum.
Arch. Microbiol. 109, 15-19.
[7] Fuchs, G., Stupperich. E. and Jaenchen, R. (1980) Autotrophic CO 2 fixation in ('hlorobium limicola. Evidence
against the operation of the Calvin Cycle in growing cells.
Arch. Microbiol. 128, 56-63.
[8] Ivanovsky, R.N., Sintsov, N.V. and Kondratieva, E.N.
(1980) ATP-linked citrate lyase activity in the green sulfur
bacterium ('hlorobium limicola forma thiosulfatophilum.
Arch. Microbiol. 128, 239-241.
[9] Barker, H.A. and Kamen, M.D. (1945) Carbon dioxide
utilization in the synthesis of acetic acid by CIostridium
thermoaceticurn. Proc. Natl. Acad. Sci. U.S.A. 31,219-225.
[10] Ljungdahl, L.G. and Wood, H.G. (1982) Acetate biosynthesis. In Vitamin Bl_,, Vol. 2 (Dolphin, D., Ed.),
Wiley, New York. pp. 166-202.
[11] Wood, H.G., Drake, H.L. and Hu, S-I. (1982) Studies with
('lostridium thermoaceticurn and the resolution of the
pathway used by acetogenic bacteria that grow on carbon
monoxide or carbon dioxide and hydrogen. Proc. Biochem. Symp., pp. 28-56. Annual Review Pasadena, CA.
[12] Wood, H.G., Ragsdale, S.W. and Pezacka, E. (1986) A
new pathway of autotrophic growth utilizing carbon
monoxide or carbon dioxide and hydrogen. Biochem. Int.,
12, 421-440.
360
[ 13] Ljungdahl, L.G. (1986) The autotrophic pathway of acetate
synthesis in acetogenic bacteria. Ann. Rev. Microbiol., in
press.
[14] Pezacka, E. and Wool, tI.G. (19841 Role of carbon monoxide dehydrogenase in the autotrophic pathway used by
acetogenic bacteria. Prec. Natl. Acad. Sci. USA 81,
6261-6265.
[15] Yagi, T. (19581 Enzymic oxidation of carbon monoxide.
Biochim. Biophys. Acta 30, 194-195.
[16] Diekert, G., Fuchs, G. and Thauer, R.K. (1985) Properties
and function of carbon monoxide dehydrogenase from
anaerobic bacteria, in Microbial Gas Metabolism: Mechanistic, Metabolic and Biotechnical Aspects (Pc×~lc, R.K.
and l)ow, C.W.. Eds.) pp. 115-130. Academic Press, New
York.
[17] Kim, Y.M. and Hegeman, (i.D. (19831 Oxidation of carbon
monoxide by bacteria. Int. Re'.'. Cytol. 81, 1-28.
[18] Ragsdale, S.W., Clark, J.E., Ljungdahl, L.G., Lundie. L.I,.
and Drake, H . L (1983) Properties of purified carbon
monoxide dehydrogenase from ('lostridtum thermoactictum, a nickel, iron sulfur protein. J. Biol. Chem. 258.
2364-2369.
[19] Meyer, O. and Rohde, M. (19841 Enzymology and bioenergetics of carbon monoxide-oxidizing bacteria, in Microbial Growth on C I Compounds (Crawford, R.I.. and
Hanson, R.S., Eds.) pp. 26-33. American St~iety for
Microbiology, Washington, DC.
[20] Hu, S-I., Drake. H.L. and W ~ d . H.(I. (19821 Synthesis of
acetyl coenzyme A from carbon monoxide, methyltetrah3;drofolate, and coenzyme A by enzymes from Clostridium
thermoaceticum. J. Bacteriol. 149, 440-448.
[21] Ragsdale, S.W. and Wood, H.G. (1985) Acetate biosynthesis by acetogenic bacteria. Evidence that carbon
monoxide dehydrogenase is the condensing enzyme that
catalyzes the final steps of synthesis. J. Biol. C h e m 260.
3970-3977.
[22] Pezacka, I 5. and Wool, II.CL (1984) The synthesis of
acetyI-CoA by Clostrldium thermoaceticum from carbon
dioxide, hydrogen, coenzyme A and methyltetrahydrofolate. Arch. Microbiol. 137, 63-69.
[231 ('lark, J.E. and Ljungdahl. L.G. (19841 Purification and
properties of 5,10-methylenetetrahydrofolate reductase, an
iron-sulfur flavoprotein from Clostridium formtcoaceticum.
J. Biol. (7'hem. 259, 10845-10849.
[24] Drake, H.I,., Hu, S-I. and Wool, H.CI. (19811 Purification
of five components from Clostridium thermoaceticum which
catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. J. Biol. Chem. 256, 11137-11144.
[25] Hu, S-I., Pezacka, E. and Wood, H.G. (19841 Acetate
synthesis from carbon monoxide by (7ostridlum thermoaceticum. Purification of the corrinoid protein. J. Biol.
('hem. 259, 8892-8897.
[26] Pezacka, E. and Wool, tt.G. (1986) The autotrophic
pathway of acetogenic bacteria. Role of CO dehydrogenase disulfide reductase. J. Biol. (?'hem., 261, 1609-1615.
[27] Pezacka, E. and W~'~d, H.G. (19861 Autotrophic growth:
methylated carbon monoxide dehydrogenase as an inter-
mediate of acetyl-CoA synthesis. Fed. Prec. Fed. Am. Soc.
Exp. Biol., in press.
[28] Ragsdale, S.W.. Ljungdahl, L.G. and DerVartanian, D.V.
(1982) i~(_- and ~qNi isotope substitutions confirm the
presence of a nickel Ill-carbon species in acetogenic CO
dehydrogenases. Biechem. Biophys. Res. Commun. 115,
658-665.
[29] Ragsdale, S.W., Wood, H.G. and Antholine, W.E. (1985)
Evidence that an iron-nickel-carbon complex is formed b)
reaction of CO with the CO dehydrogenase from
('lostrtdlum thermoaceticum. Proc. Natl. Acad. Sci. USA
82, 6811 6814.
[30] Bowien, B. and Schlegel, H.G. (19811 Physiology and
biochemistry of aerobic hydrogen-oxidizing bacteria.
Annu. Rev. Microbiol. 35, ,:105 452.
[31] Zeikus, J. (1983) Metabolism of one-carbon compounds
by chemotrophic anaerobes. Adv. Microbiol. Physiol. 24,
215. 299.
[32] Barker, H.A.. Ruben, S. and Beck, J.V. (19401 Radioactive
carbon as an indicator of carbon dioxide reduction, IV.
"['he synthesis of acetic acid from carbon dioxide by
Clostridium actdt-urtci. Prec. Natl. Acad. Sci. USA 26,
477 -482.
[33] Barker, H.A. and Elsden, S.R. (19471 ('arhon dioxide
utilization in the formation of glycine and acetic acid. J.
Biol. Chem. 167, 619-620.
[34] Barker, H.A., Volcani, B.E. and Cardon, B.P. (19481 Tracer
studies on the mechanism of glycine fermentation by
Dtplococctt~" glycinophilus. J. Biol. Chem. 173, 803-804.
[35] Waber, L.J. and Wcx)d, H.G. (1979) Mechanism of acetate
synthesis from CO~ by Clostridium acldturtci. J. Bacteriol.
140, 468- 478.
[36] Di,irre, P. and Andreesen, J.R. (1982) Selenium-dependent
growth and glycine fermentation by Ch~stridium purinoh'twum. J. Gen. Microbiol. 128. 1457 1466.
1371 l)i~rre, P. and Andreesen, J.R. (1982) Pathway of carbon
dioxide reduction to acetate without a net energy requirement in ('h~stridium purinolvticum. FEMS Microbiol. Lctt.
15, 51-56.
[38] Diarre, P., Spahr, R. and Andreesen. J.R. (1983) Glycinc
fermentation via a glycine reduetase in PeTtococcus
glvctnophilus and Peptococcus m a g n ~ . Arch. Microbiol.
134, 127 135.
[39] Tanner, R.S., Wolfe, R.S.. and Ljungdahl, LG. (1978)
Tetrahydrofolate enzyme levels in Acetobacterium woodti
and their implication in the synthesis of acetate from
(?02. J. Bacteriol. 134, 668-670.
[401 Ragsdale. S.W., Ljungdahl. L.G., DerVartanian, D.V.
(1983) Isolation of carbon monoxide dehydrogenase from
Acetobactertum woodii and comparison of its properties
with those of the CIo.gtrtdtt~m thermoacettcum enzyme. J.
Bacteriol. 155, 1224-1237.
[41] Ragsdale, S.W. (1983) Electron transfer reactions involved
in acetate biosynthesis in acetogenic bacteria. Ph.D. Thesis, University of Georgia, Athens. GA.
[42] Eden, G. and Fuchs, G. (19821 Total synthesis of acetyl
361
[431
[44]
[45]
146]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
coenzyme A involved in autotrophic C() 2 fixation in
Acetohacteriurn woodii. Arch. Microbiol. 133, 66-74.
Eden, G. and Fuchs, G. (19831 Autotrophic CO, fixation
in A('etobacterium woodii. II. Demonstration of enzymes
involved. Arch. Microbiol. 135, 68-73.
I)iekert, G. and Ritter, M. (1983) Carbon monoxide fixation into the carboxyl group of acetate during growth of
Acetobacter~um woodJt on H 2 and CO 2. FEMS Microbiol.
Lett. 17. 299-302.
Kerby. R.. Niemczura, W. and Zeikus, J.G. (1983) Singlecarbon metabolism in acetogens: Analysis of carbon flow
in Acetobacterium woodii and Butyribacterium metl~vlotrophicum by fermentation and ~(" nuclear magnetic
resonance measurement. J. Bacteriol. 155. 1208-1218.
Diekert. G.B. and Thauer, R.K. (19781 Carbon monoxide
oxidation by Clostrtdtum thermoaceticum and Clostridturn
formu'oaceticum. J. Bacteriol. 136. 597-606.
Diekert, G., Hansch. M. and Conrad, R. (1984) Acetate
synthesis from 2 C(), in acetogenic bacteria: is carbon
monoxide an intermediate? Arch. Microbiol. 138. 224-228.
Clark. J.E.. Ragsdale, S.W., Ljungdahl. L.G. and Wiegel.
J. (19821 Levels of enzymes involved in the synthesis of
acetate from CO, in Clostridium thermoautotrophicum. J.
Bacteriol. 151,507-509.
l)aniels. I,.. Sparling. R. and Sprott. G.D. (1984) The
bioenergetics of methanogenesis. Biochemi. Biophys. Acta
768. 113-163.
Wolfe, R.S. (1985) Unusual coenzymes of methanogenesis.
Trends Biochem. Sci. 10, 396- 399.
Daniels, L. and Zeikus, J.G. (1978) One-carbon metabolism in methanogenic bacteria: Analysis of short term
fixation products of 14CO 2 and 14CH~OH incorporated
into whole cells. J. Bacteriol. 136, 75-84.
Fuchs, G. and Stupperich. E. (1978) Evidence for an
incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophwum. Arch. Microbiol. 118,
121-125.
Fuchs. G. and Stupperich, E. (19801 Acetyl-CoA, a central
intermediate of autotrophic CO, fixation in Methanobucteriurn thermoautotrophicum.
Arch. Microhiol. 127,
267-272.
Stupperich, E. and Fuchs. G. (1984) Autotrophic synthesis
of activated acetic acid from two CO, in Methanobacterium thermoautotrophicum, If. Evidence for different
origin of acetate carbon atoms. Arch. Microbiol. 139,
14- 20.
Ruhlemann, M.. Ziegler. K.. Stupperich, E. and Fuchs. G.
(19851 Detection of acetyl coenzyme A as an early CO 2
assimilation intermediate in Methanohactertum. Arch. Microbiol. 141,399-406.
Evans, J.N.S.. Tolman, C.J. and Roberts, M.F. (1986)
Indirect observation by 13C NMR spectroscopy of a novel
CO, fixation pathway in methanogens, Science 231,
488-491.
Lange. S. and Fuchs, G. (19851 Tetrahydromethopterin, a
ct~enzyme involved in autotrophic acetyl coenzyme A
synthesis from 2 CO2 in Methanobacterium. FEBS Lett.
181,303--307.
[58] Leigh. J.A., Rinehart. K.L.. Jr. and Wolfe, R.S. (19851
Methanofuran (carbon dioxide reduction factor), a formvl
carrier in methane production from carbon dioxide in
Methanohacterium. Biochemistr)' 24. 995. 999.
[59] van Beelen. P.. van Neck, J.W.. de Cock, R.M., Vogels,
G.D., Guijt, W. and Haasn(x)t, C.A.G. (19841 5,10methenyl-5.6.7,8-tetrahydromethanopterin,
a one-carbon
carrier in the prcx:ess of methanogenesis. Bit,chemistry 23.
4448-4454.
[60] Taylor, C.I). and Wolfe. R.S. 119741 Structure and methvlation of co.enzyme M (HS('H 2('H2SO~). J. Biol. ('hem.
249, 4879-4885.
[61] Eirich, LD., Vogels, G.D., and Wolfe. R.S. (19781 Proposed structure of coenzyme k'42¢~ from methanobacterium. Biochemistry 17, 4583-4593.
[62] Pfaltz, A., Juan, B.. Fassler. A., Eschenmocer, A.,
Jaenchen, P., Gilles, H.H.. Diekert, G. and Thauer, R.K.
(19821 Zur Kenntnis des Factors F4~o aus methanogenen
Bakterien: Structur des porphinoiden Ligen Systems. itelv.
('him. Acta 65, 828-865.
[63] Leigh. J.A. (19831 Levels of water-soluble vitamins in
methanogenic and non-methanogenic bacteria. Appl. Environ. Microbiol. 45. 800-803.
[64] Stupperich, E. and Fuchs. G. (1984) Autotrophic synthesis
of activated acetic acid from 2 CO~ in Methanobaeterium
thermoautotropwum, I. Properties of in vitro system. Arch.
Microbiol. 139. 8 13.
[65] Romesser. J.A. and Wolfe, R.S. (1982) Coupling of methyl
coenzyme M reduction with carbon dioxide activation in
extracts of Methanohacterium thermoautotrophicum. J.
Bacteriol. 152. 840-847.
[66] Taylor. R.T., Whitfield, C. and Weissbach. H. (19681
Chemical propylation of vitamin-B~2 transmethylase:
Anomalous behavior of S-adenosyl-L-methionine. Arch.
Biochem. Biophys. 125, 240-252.
[67] Holder, U., Schmidt, D-E., Stupperich, E. and Fuchs. G.
(1985) Autotrophic synthesis of activated acetic acid from
two ('O2 in Methanobacterium thermoautotrophicum, Ill.
Evidence for common one-carbon precursor pool and the
role of corrinoid. Arch. Microbiol. 141,229-238.
[68] Evans, J.N.S., Tolman, C.J., Kanodia, S. and Roberts.
M.F. (19851 2,3-cyclopyrophosphoglycerate in methanogenesis: Evidence by 13(- NMR spectroscopy for a role in
carbohydrate metabolism Biochemistry 24, 5693-5698.
[69] Blaylock. B.A. and Stadtman. T.C. (19641 Enzymic formation of methylcobalamin in Methanosarcinu barkert extracts. Biochem. Biophys. Res. Commun. 17. 475-480.
[70] Blaylc~zk, B.A. (1968) Cobamide-dependent methanolcyanocob(1)alamin methyltransferase of Methanosarcinu
barkert. Arch. Biochem Biophys. 124, 314-324.
[71] Taylor, C.I). and Wolf, R.S. (19741 A simplified assay for
coenzyme M (HSCH zCH2SO3). Resolution of methylcobalamin-coenzyme M methyltransferase and use of
sodium borohydride. J. Biol. Chem. 249. 4886-4890.
[72] Wo(xl, J.M., Moura, I., Moura, J.J.G.. Santos, M.H.,
Xavier. A.V.. LeGall, J. and Scandellari. M. (19821 Role
of vitamin B~2 in methyl transfer for methane biosynthesis bv Methanosarcma harkeri. ,~ience 216, 303-305.
362
[731 van der Meijden, P., the Brrmmelstroet, B.W., Poirot,
C.M., van der Drift, C. and Vogels, G.D. (1984) Purification of properties of methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarctna
barkeri. J. Bacteriol. 160 629-635.
[74] van der Meijden, P., Heythuysen. H.J., Pouwels, A.,
Houwen, F., van der Drift, C. and Vogels, G.D. (1983)
Methyltransferases involved in methanol conversion by
Methanosarcina barkeri. Arch. Microbiol. 134, 238-242.
[75] van der Meijden, P. (1984) Methanol conversion by
methanogenic acetogenic bacteria. Thesis, University of
Nijmegen, The Netherlands.
[76] Eikmanns. B. and Thauer, R.K. (1985) Evidence for the
involvement and role of a corrinoid enzyme in methane
formation from acetate in Methanosarcina harkeri. Arch.
Microbiol. 142, 175-179.
[77] Kenealy, W.R. and Zeikus, J.G. (1982) One-carbon
metabolism in methanogens: Evidence for the synthesis of
a two-carbon cellular intermediate and unification of
catabolism and anabolism in Methanosarcina barkerl. J.
Bacteriol. 151,932-941.
[78] Shapiro, S. (1982) Do corrinoids function in the methanogenic dissimilation of methanol by Methanosarcma
barkeri? Can. J. Microbiol. 28, 629-635.
[79] Whitman, W. and Wolfe, R.S. (1985) Activation of the
methylreductase system from Methanobacterium b~'antit
by corrins. J. Bacteriol. 163, 165-172.
[80] Thauer, R.K., Fuchs, G., Kaufer, B. and Schnitker, U.
(1974) Carbon monoxide oxidation in cell-free extracts of
Clostridium pasteurianum. Eur. J. Biochem. 45, 343-349.
[81] Krzycki, J.A. and Zeikus, J.G. (1984) Characterization
and purification of carbon monoxide dehydrogenase from
Methanosarcina barkeri. J. Bacteriol. 158, 231-237.
[82] Zeikus, J.G.. Kerby, R. and Krzycki, J.A. (1985) Singlecarbon chemistry of acetogenic and methanogenic bacteria.
Science 227, 1167-1173.
[83] Krzycki, J.A., Wolkin, R.H. and Zeikus, J.G. (1982) Comparison of unitrophic and mixotropbic substrate metabolism by an acetate adapted strain of Methanosarcina
barkeri. J. Bacteriol. 149, 247-254.
[84] Krzycki, J.A. and Zeikus, J.G. (1984) Acetate catabolism
by Methanosarcina barkeri: hydrogen-dependent methane
production from acetate by a soluble protein fraction.
FEMS Microbiol. Lett. 25, 27-32.
[85] Ivey, D.M. and Ljungdahl. L.(L (1986) Purification and
characterization of the F1-ATPase from Clostridiurn therrnoaceticum. J. Bacteriol. 165, 252-257.
[86] Krzycki, J.A., Lehman, L.J. and Zeikus, J.G. (1985)
Acetate catabolism by Methanosarcina barker~: evidence
for involvement of carbon monoxide dehydrogenase,
methyl coenzyme M, and methylreductase. J. Bacteriol.
163, 1000-1006.
[87] Baresi. L. (1984) Methanogenic cleavage of acetate by
lysates of Methanosarcma barkeri. J. Bacteriol. 160,
365- 370.
[88] Eikmanns, B. and Thauer, R.K. (1984) Catalysis of an
isotopic exchange between CO 2 and the carboxyl group of
acetate by Methanosarcina barkeri grown on acetate. Arch.
Microbiol. 138, 365-370.
1891 Widdel, F. (1983) Methods for enrichment and pure culture isolation of filamentous gliding sulfate reducing
bacteria. Arch. Microbiol. 134, 282-285.
[90] Widdel, F., Kohring, G-W., and Mayer, F. (1983) Studies
on dissimilatory sulfate-reducing bacteria that decompose
fatty acids, III. Characterization of the filamentous gliding Desulfimema hmicola gen. nov. sp. nov., and Desulfonema magnum sp. nov. Arch. Microbiol. 134. 286-294.
[91] Klemps, R.. Cypionka, H.. Widdel, F., and Pfennig, N.
(1985) Growth with hydrogen, and further physiological
characteristics of Desulfotomaculum species. Arch. Microbiol. 143, 203-208.
[921 Jansen. K., Thauer, R.K, Widdel, F. and Fuchs, G. (1984)
Carbon assimilation pathways in sulfate-reducing bacteria.
Formate, carbon dioxide, carbon monoxide and acetate
assimilation by Desulfot,ibrlo baarsii. Arch. Microbiol. 138,
257-262.
[93] Vogels, G.D. (1979) The global cycle of methane. Antonie
van Leeuwenhoek 45. 347-352.
[94] Bott, M.H., Eikmanns. B., and Thauer, R.K. (1985)
Defective formation and/or utilization of carbon monoxide in H 2 / C O 2 fermenting metanogens dependent on
acetate as carbon source. Arch. Microbiol. 143. 266-269.
[951 Wood. H.G. and Barden, R.E. (1977) Biotin enzymes.
Ann. Rev. Biochem. 46, 385-413.