FEMS MicrobiologyReviews87 (1990) 373-376
Published by Elsevier
373
FEMSRE 00188
Sodium bioenergetics in methanogens and acetogens
Volker MUller *, M i c h a e l Blaut, R e n o Heise, Christiane W i n n e r a n d G e r h a r d G o t t s c h a l k
lnstitutf~r Mikrobiologie,G~ttingen,F.R.G.
Key words: Metha~-togenesis; Acetogenesis; Na + gradients; N a + / H + antiport; Reverse electron transport:
Sodium-motive redox systems
1. S U M M A R Y
Recent investigations with Methanosarcina
barkeri elucidated the role of sodium ions in the
energy metabolism of methanogenic bacteria and
provided evidence for a novel mechanism of energy transduction with Na + as the coupling ion.
During methanogenesis from methanol, an
cletrochemical sodium gradient generated by a
N a + / H + antiporter is used as the driving force
for the thermodynamically unfavourable oxidation
of methanol to the formal redox level of formaldehyde. During methanogenesis from H 2 +
CO 2, the reverse reaction, the reduction of formaldehyde to the level of methanol, is accompanied by a primary, electron transport-driven
sodium extrusion. Acetogen~sis from H 2 + CO 2 as
carried out by Acetobacterium woodii is a sodiumdependent process and is accompanied by the
generation of a transmembrane sodium gradient
with the reduction of formaldehyde to the level of
methanol as the sodium-dependent step.
Correspondence to: G. Gottschalk, Institut ffir Mikrobiologie,
Grisebachstrasse 8,1)-3400 Ggningen, F.R.G.
. Present address: Yale University,Department of Molecular
Biophysicsand Biochemistry,New Haven, CT 06511, U.S.A.
2. I N T R O D U C T I O N
Whereas it is known for some time now that
methanogenesis as well as growth of methanogenic
bacteria on various substrates is strictly dependent
on the presence of sodium ions h,. the medium [1],
a sodium dependence of acetate formation was
described only recently [2,11]. In analogy to other
systems a possible role of Na + in intracellular
enzyme catalysis, transport processes and the
mechanism of ATP synthesis was discussed for
methanogens. However, experimental data concerning the magnitude of the electrochemical
sodium gradient and the mechanism for its generation as well as its utilization became only available a few years ago. We briefly summarize here
the results which led to the discovery of a reversible, sodium-motive electron transport chain in
methanogens and its possible analogy in acetogens.
3. RESULTS A N D C O N C L U S I O N S
3.1. The sodium cycle in methylotrophic methanogens: a secondary sodium ion gradient generated by
a Na + / H + antiporter as the driving force for
reverse electron transport
The first clue for the role of sodium ions in the
path of methanogenesis came from a study using
resting cells of M. barkeri with methanol as a
0168-6445/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
374
substrate. Methanol is disproportionated to
methane and carbon dioxide according to (equations 1-3):
CH3OH + H20 --* CO 2 + 6H
(1)
3 CH3OH + 6H --' 3CH4 + 3H20
4 CH3OH --* 3 CH4 + CO2 + 2 H20
(2)
(3)
By comparing the effect of Na + on methanogenesis from different substrates and substrate
combinations it was shown that sodium ions are
neither involved in the final step of methanogenesis, the reduction of methyl-CoM to methane, nor
in the mechanism of methanogenesis-driven ATP
synthesis [3]. The latter finding is supported by
the inability to demonstrate ApNa-driven ATP
synthesis by applying Na + pulses to resting cells
pregrown on methanol [4] as well as measuring the
Na + flux across the membrane in the presence or
absence of uncouplers [5]. In addition, the ATPase
activity in membrane preparations is not stimulated by Na + [6].
In the presence of methanol and formaldehyde
as substrate.,., ,'aethanogenesis takes place even in
the absence of Ha + with formaldehyde exclusively
oxidized to CO2 and methanol reduced to
methane, indicating that the oxidation of methanol
to the formal redox level of formaldehyde is the
sodium-dependent reaction in this pathway. Interestifigly, this reaction is also uncoupler-sensitive
[3]. According to the experimental data, and taking into account thermodynamic considerations,
the need of a reverse electron transport coupled to
this reaction is strongly suggested: electrons derived from tI~e redox couple CH3OH/HCHO (E~
= - 1 8 2 mV) have to be transported in an
energy-dependent manner to the level of H 2 or
F,n0 (E~ffiffi- 4 2 0 mV and E ~ = - 3 4 0 to - 3 5 0
mV, respectively) in order to be channelled into
the methylreductase reaction.
That the energy needed for this reaction is
provided by A~Na+ was concluded from the following results [7]: 1. Methanogenesis from
methanol or methanol + H 2 is accompanied by
the generation of a secondary sodium ion gradient
via a harmaline- and amiloride-sensitive N a + / H +
antiporter which is energized by the primary electrochemical proton gradient [5]. 2. Inhibition of
the N a + / H + antiporter leads to an inhibition of
methanogenesis from methanol (Eqn. 3) but not
from methanol + H2 (Eqn. 2). 3. Methanogenesis
is impaired by artificially inversed sodium gradients (Na + > Na+). 4. Dissipation of A/~Na+ leads
to an inhibition of methanogenesis even if A/~,+
is high. These experiments demonstrate that not
the sodium ion per se but an electrochemical
sodium gradient is required for methanol oxidation.
3.2. The sodium cycle in autotrophic methanogens:
generation of a primary electrochemical sodium
gradient via electron transport-driven sodium extrusion
The obtervation that the oxidation of methanol
to the redox level of formaldehyde is driven by
A#Na+ led to the idea that the reverse reaction
may be coupled to a primary, electron transportdriven Na + extrusion.
Experiments with resting cells of M. barkeri
showed that the reduction of CO 2 is accompanied
by the generation of a transmembrane Na + gradient and that the reduction of formaldehyde to the
level of methanol is the sodium-dependent step
[5]. That this reaction is indeed coupled to a
primary, electron transport-driven sodium pump
was concluded from the following results [5]: 1.
Methanogenesis from H 2 + HCHO is accompanied by the generation of a transmembrane Na +
gradient. 2. The substrate-dependent efflux of Na +
is not impaired by inhibition of the N a + / H +
antiporter or by uncouplers. 3. Uncouplers do not
affect the Ag, but lead to the generation of a ApH
of the same value but opposite in polarity (inside
acidic). 4. The presence of ATPase inhibitors has
no effect on the generation of A/tNa+. These experiments demonstrate the presence of a primary
sodium pump, driven by electron transport from
H 2 to methylene-tetrahydromethanopterin (H 4
MPT) or by a hypothetic electron transport coupled to the methyl transfer from methyl-H 4 MFI'
to coenzyme M. This is the first example of a
sodium-motive redox chain in an anaerobic
bacterium. The role of sodum ions in the path of
methanogenesis is depicted in Fig. 1.
The electrochemical sodium gradient is used for
different purposes. In Methanococcus voltae, a
3"15
/
,.~X-I'h
I
[-~
2tr
+.~i+
I
I
-++
t °+T
\ ,,...~
l't +
.../]
kNa +
Fig. I, Role of sodium ions in the path of methanosenesis from
H 2 +CO 2 (
) and methanol ( . . . . . . ). It is not yet clear
whether methanolenters the pathway at the level of methylCoMor methyI-H4MPT.
Na+/amino acid symport has been described for
the transport of isoleucin [8], and for Methanebacterium thermoautotrophicum a role of Na + in
the regulation of the intracellular pH via the
N a + / H + antiporter has been postulated [9]. Regarding the energetics of the first reduction step of
CO 2 to formylmethanofuran, another possible
function of A/tr4,+ was envisaged: this reaction is
thermodynamically unfavourable and, therefore, a
coupling with the exergonic reduction of formaldehyde to the level of methanol was postulated
[4].
313. Sodium bioenergetics in acetogens
During acetogenesis from Hz + CO, the CImoiety is converted to acetate via formyl- methenyi- and methylene- to methyl-tetrahydrofolate
(H4F); the formation of formyl-H4F requires the
hydrolysis of 1 ATE Methyl-H4F is condensed
with a bound CO, derived from the reduction of a
second molecule of CO2, to yield acetybCoA. The
subsequent acetate formation via acetyl phosphate
then yields 1 ATP by substrate level phosphorylation [10]. The ATP balance from substrate level
phosphorylation is therefore zero and an additional mechanism for the conservation of ATP
must be present.
The discovery of a primary electrochemical
sodium gradient generated during the reduction of
formaldehyde in methanogens prompted a study
to determine and characterize the sodium dependence of acetogenesis using Acetobacterium woodii
as a model organism. Growth as well as acetogenesis from fructose is only sfightly dependent on
sodium ions, whereas growth on methanol or Hz
+ COz as well as acetate formation is strictly
dependent on Na + [11]. The sodium-dependent
step during acetogenesis from H 2 + CO2 was
eiucidated using different substrates and substrate
combination3: wh~rca~ acetate formation from H 2
+ COz and H 2 + HCHO + CO was sodium-dependent, acetogenesis from H e + methanol + CO
was not [11]. These results indicated that the reduction of methylene- to methyl-H+F or the subsequent methyl transfer from methyl-H4F to COdehydrogenase is a sodium-dependent step in the
path of acetogenesis from H 2 + COz. A sodium
dependence was also observed for acetogenesis
from CO + CO e as carried out by Peptostreptococcus productus [2].
Acetate formation from H a + CO 2 by A. woodii
is accompanied by the generation of a rather steep
transmembrane Na + gradient in the range of - 9 0
mV, which can be dissipated by monensin. Interestingly, addition of monensin to resting cells incubated in the presence of low Na + stimulates
acetogenesis from H 2 + HCHO + CO [11]. This
behaviour resembles 'respiratory control' and can
be taken as an indication for a sodium-motive
redox system connected to the reductase or methyl
transferase reaction. The corresponding reductase
was recently shown to be membrane bound [12],
which is in accordance with its possible function
as a primary sodium pump. It is important to
mention that this reaction is the only one exergonic enough to be coupled to ATP synthesis
during acetogenesis from H2+CO,. and was
376
therefore considered to b e involved in energy conservation [13].
N o data concerning the function o f the sodium
gradient are available b u t it could b e used as a
driving force for endergonic redox reactions, p H
regulation u n d e r acidic conditions, t r a n s p o r t
processes or A T P synthesis. F o r the latter, two
mechanisms are conceivable: A / ~ a ÷ is the driving
force for A T P synthesis via a Na+-translocating
A T P a s e o r A/~Na÷ is converted to a s e c o n d a r y
proton potential which gives rise to A T P synthesis
via a H+-translocating ATPase.
3. 4. Concluding remarks
T h e discovery o f a reversible sodium-motive
redox system in m e t h a n o g e n s a n d its possible
analogy in acetogens readily explains the sodium
d e p e n d e n c e o f growth as well as p r o d u c t formation from different substrates, It also provides a
n e w m e c h a n i s m for energy conservation a n d energy transduction in strictly anaerobic bacteria. It
is remarkable that Methanosarcina barkeri extrudes p r o t o n s as well as sodium ions during the
fermentation o f H 2 + C O 2 by m e a n s o f an elect r o n transport system. W h y m e t h a n o g e n s establish
p r i m a r y p r o t o n as well as sodium gradients can
n o t b e answered at the m o m e n t .
ACKNOWLEDGEMENTS
T h e work f r o m the laboratory of the authors
was s u p p o r t e d b y the D e u t s c h e Forschungsgemeinschaft.
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