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Journal of Bacteriology, April 2000, p. 2200-2206, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence for the Presence of an F-Type ATP Synthase
Involved in Sulfate Respiration in Desulfovibrio
vulgaris
Kiyoshi
Ozawa,1
Takanori
Meikari,1
Ken
Motohashi,2
Masasuke
Yoshida,2 and
Hideo
Akutsu1,*
Department of Chemistry and Biotechnology,
Faculty of Engineering, Yokohama National University, Hodogaya-ku,
Yokohama 240-8501,1 and Research
Laboratory of Resources Utilization, R-1, Tokyo Institute of
Technology, Nagatsuta 4259, Yokohama 226-0026,2
Japan
Received 2 November 1999/Accepted 1 February 2000
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ABSTRACT |
Using a library of genomic DNA from Desulfovibrio
vulgaris Miyazaki F, a strict anaerobe, and two synthetic
deoxyoligonucleotide probes designed for F-type ATPases, the genes for
open reading frames (ORFs) 1 to 5 were cloned and sequenced. The
predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and
that they share considerable identity at the amino acid level with 
,
, 
, 
, and 
subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was
partially purified from the cytoplasmic membrane fraction of the
D. vulgaris Miyazaki F cells. The N-terminal amino
acid sequences of three major polypeptides separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis were identical to
those of the products predicted by the sequences of ORF-2, ORF-3, and
ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the
F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured
aerobically. It indicates that the enzyme works as an ATP synthase in
the D. vulgaris Miyazaki F cells in connection with sulfate respiration.
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INTRODUCTION |
The ATPases have been classified
into three major groups, the F, V, and P types (25). F-type
ATPases are the most common H+-translocating ATP
synthases. They consist of two parts, namely Fo, a
membrane-embedded portion, and F1, a soluble domain. The Fo portion generally serves as a proton channel, but in
some cases it may also serve for the transport of Na+
(10). The F1 portion is composed of five
subunits, i.e., , , , , and , with stoichiometry of
3:3:1:1:1. The genes encoding these proteins form a cluster, not only
in aerobic and anaerobic eubacteria but also in archaebacteria
(30). F-type ATPases are located in the membranes of
bacteria, chloroplasts, and mitochondria and catalyze the hydrolysis or
synthesis of ATP coupling with H+ (or Na+)
transport across a membrane. It is accepted that aerobic organisms use
the enzyme mainly for synthesizing ATP; e.g., the enzyme functions as
an ATP synthase. In contrast, this is not yet well established for
strict anaerobes, which are unable to utilize molecular oxygen as a
terminal electron acceptor. It has been anticipated that F-type
ATPases usually hydrolyze ATP to pump out H+ in strict
anaerobes. However, Propionigenium modestum (11) and Acetobacterium woodii (27), strictly
anaerobic bacteria, have Na+-translocating ATP synthases,
which synthesize ATP. The generation of an Na+ gradient in
P. modestum is coupled to a decarboxylation reaction by
Na+-translocating membrane-bound methylmalonyl-coenzyme A
(CoA) decarboxylase (11). Moorella thermoacetica
(the former name was Clostridium thermoaceticum
[5]), which is also an obligatory anaerobic bacterium,
was indicated to have a H+-translocating ATP synthase
(13). This is thought to synthesize ATP by using the
H+ gradient generated by an anaerobic electron transport
that involves electron carriers, such as cytochromes
b554 and b559,
menaquinone, rubredoxin, ferredoxin, and a flavoprotein, although the
ultimate electron acceptor of the electron transport chain has not been identified yet.
Gram-negative eubacterium Desulfovibrio vulgaris Miyazaki F
is a strict anaerobe that uses sulfate as a terminal electron acceptor.
While electron transport proteins involved in the energy transduction
in the sulfate-reducing bacteria have been extensively investigated
(24), little is known about the ATPase (4,
18). Although D. vulgaris produces 2 mol of ATP
through the oxidation of 2 mol of lactate to acetic acid, they are used
out for the formation of adenosine 5'-phosphosulfate during the
reduction of a sulfate ion (20). Thus, for living activity,
the bacterium should have other ATP-generating systems. One of the
possible sources is an energy conversion system coupled to the electron transport. Odom and Peck proposed a chemiosmotic hydrogen cyclic model
as a general mechanism for energy coupling in Desulfovibrio species (21). It predicts generation of the proton gradient through the oxidation of hydrogen molecules coupled to the sulfate reduction. Actually, an increase of the proton concentration in the
bulk phase was observed as a function of hydrogen consumption (4,
17). However, there has been no report on the identification of a
H+-translocating ATP synthase in Desulfovibrio.
This enzyme has a twofold importance in Desulfovibrio,
namely as a key enzyme in the energy conversion system and as a
milestone in the evolution of the biological energy conversion systems.
In this study, we report the cloning and sequencing of the genes of the
, , , , and subunits of F1-ATPase and a
partial purification of the enzyme, which confirm the presence of
F-type ATPase in D. vulgaris Miyazaki F for the first time.
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MATERIALS AND METHODS |
Materials and bacterial strains.
The pUC118 vector,
restriction enzymes, modifying enzymes, and Ex Taq
polymerase were purchased from Takara Shuzo Co., Ltd. (Kusatsu, Japan).
Radiochemicals [-35S]dCTP (specific radioactivity, 400 Ci mmol
1, 10 mCi ml1) and
[-32P]dATP (3,000 Ci mmol1, 10 mCi
ml1) were purchased from Amersham Pharmacia Biotech
(Uppsala, Sweden) and were used for dideoxynucleotide
sequencing and 5'-end labeling, respectively. Synthetic
oligonucleotides, Hybond-N+ filters, Thermo Sequenase cycle
sequencing kits, a MonoQ column (5-mm inner diameter by 5 cm), and
phenyl Sepharose resin were also obtained from Amersham Pharmacia
Biotech. Deoxyribonuclease I (DNase I) and phenylmethylsulfonyl
fluoride (PMSF) were from Sigma Chemical Co. (St. Louis, Mo.). The
vector 
EMBL3, Gigapack II gold packaging extracts, Escherichia
coli JM109, and XL1-Blue MRA(P2) were obtained from Toyobo Co.,
Ltd. (Osaka, Japan). All other reagent-grade chemicals and antibiotics
were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
D. vulgaris Miyazaki F cells were grown in the Postgate C
medium (26) at 37°C.
Construction of a genomic library of D. vulgaris
Miyazaki F.
D. vulgaris Miyazaki F cells were lysed with
0.5% (wt/vol) sodium dodecyl sulfate (SDS) and treated with 100 µg
of proteinase K per ml. After cell wall debris, polysaccharides, and
remaining proteins were removed by selective precipitation with CTAB
(cethyltrimethylammonium bromide) (19), the genomic DNA was
recovered from the resulting supernatant by isopropanol precipitation.
Two deoxyoligonucleotide primers for PCR were designed according to the
known amino acid sequences of the subunits of
F1-ATPases (F1). Primer 1 was a 23-mer
with the nucleotide sequence 5'-GGCGGCGCGGGCGTGGGCAAGAC-3' (corresponding to the amino acid sequence of
G-G-A-G-V-G-K-T-V in the so-called P-loop region), while primer 2 was a
27-mer with the nucleotide sequence
5'-GTCGGTAAGGTCGTCCGCGGGCACGTA-3'. This is complementary
to the base sequence corresponding to the amino acid sequence of
Y-V-P-A-D-D-L-T-D in the central domain. The known codon preference
caused by the high GC content (65%) of D. vulgaris DNA
(26) was taken into account in the design of these
deoxyoligonucleotides. About 500-bp DNA fragments were amplified from
the D. vulgaris Miyazaki F genomic DNA by PCR. The amplified 500-bp DNA fragments were phosphorylated with T4 polynucleotide kinase
and ligated with pUC19 vector previously cut with SmaI. The
resultant plasmid was sequenced completely by using two universal primers, 5'-CAGGAAACAGCTATGAC-3' and
5'-GTTTTCCCAGTCACGAC-3'. On the basis of the DNA sequence
obtained, four kinds of new sequencing primers (primers 3, 4, 5, and 6)
for the F1 gene (atpD) were synthesized.
Their sequences are 5'-CCTTGGTGGCCTGCAGGAACGCATC-3' (25-mer), 5'-GATGCGTTCCTGCAGGCCACCAAGG-3' (25-mer),
5'-GGTGTTGGCGAGCGTACCCG-3' (20-mer), and
5'-CGGGTACGCTCGCCAACACC-3' (20-mer), respectively. These
oligonucleotides were also used for the hybridization experiments described below.
For the preparation of genomic library, the genomic DNA was partially
digested with Sau3AI. The digested fragments were then subjected to size fractionation via isopycnic centrifugation at 140,000 × g for 24 h by using a 5 mM EDTA-20 mM
Tris-HCl buffer (pH 8.0) containing 40% (wt/vol) sucrose. The
fractions containing 15 to 20 kbp of DNA fragments were diluted
threefold with a 1 mM EDTA-10 mM Tris-HCl buffer (pH 8.0) and
collected by ethanol precipitation. These 15- to 20-kbp DNA fragments
were ligated into the vector EMBL3, which had been cut with
BamHI previously. The ligated DNA was then reconstituted
into phage by using Gigapack II gold packaging extracts and were
transfected to E. coli XL1-Blue MRA(P2). This strain allows
the transfection of only recombinant phages by Spi/P2 selection
(12). The obtained phages with the D. vulgaris Miyazaki F genomic library were amplified to
approximately 108 PFU as stocks.
Cloning and dideoxy sequencing.
The plaques carrying 15- to
20-kbp DNA fragments of the D. vulgaris Miyazaki F genomic
library were blotted onto Hybond-N+. It was found by
autoradiography that 34 per 1,000 plaques hybridize with primer-3 and
primer-5. A phage stock was prepared from a positive plaque, and the
hybridization with the primers was reconfirmed. Then, the phage stock
was amplified to approximately 108 PFU.
The
phage DNA was isolated from the phage lysate by the Qiagen
lambda starter kit (Funakoshi, Tokyo, Japan). The purified
DNA
(
MK27, hereafter) contained a 17-kbp insert. It was digested
with
either
SalI,
PstI, or both
PstI and
SphI, and then separated
electrophoretically in a 0.7%
agarose gel. The separated fragments
were subcloned into the same site
of pUC118 vectors. These recombinant
plasmids were designated pMK1, -2, -3, and -4, respectively. The
pMK1 and pMK2 plasmids contained
SalI inserts of 4,700 and 750
bp, respectively, and pMK3 and
pMK4 contained a
PstI insert of
3,000 bp and a
PstI-
SphI insert of 1,200 bp, respectively. The
pMK1 vector was then digested with both
SphI and
SalI, and the
resultant
SphI-
SalI
fragment (900 bp) was resubcloned into pUC118.
This pMK1 derivative
vector was designated pMK11. Furthermore,
the
SalI insert of
4,700 bp in pMK1 was digested with
Sau3AI and
the resultant
660-bp fragment was resubcloned into a
BamHI site
of pUC118.
This pMK1 derivative vector was designated pMK12. Deletion
mutants of
the pMK1 insert in different lengths were also obtained
by using
exonuclease III and Mung bean nuclease. The pMK2 vector
was digested
with
PstI, or with both
PstI and
SalI,
and the resultant
three fragments were individually subcloned into
pUC118, yielding
pMK21 (100-bp insert), pMK22 (250-bp insert), and
pMK23 (400-bp
insert). All plasmids mentioned above were subjected to
restriction
mapping. The coding regions of these plasmid inserts were
sequenced
on both strands, and each base was completely determined by
the
dideoxy chain termination method (
28). The plasmids used
in
this work are summarized in Table
1.
Purification of F1-ATPase from D. vulgaris Miyazaki F.
Preparation of cytoplasmic membrane was
previously described (23). A 3-ml suspension of cytoplasmic
membrane (15 mg of protein/ml) was mixed with an equal volume of
chloroform. This mixture was vigorously shaken for 2 min and
centrifuged in six Eppendorf tubes at 15,000 rpm at 4°C for 15 min by
using a Tomy MRX-150 microcentrifuge. The clear supernatant was diluted
10 times in ultrapure water and applied on a MonoQ column (5-mm inner
diameter by 5 cm) equilibrated with a 1 mM EDTA-20 mM Tris-HCl (pH
8.0) solution (buffer A). Fractions containing ATPase activity were
obtained by elution of buffer A with a linear NaCl gradient (0 to 0.5 M). Solid ammonium sulfate was added to the collected fraction. The
final ammonium sulfate concentration was 30% (wt/wt). After
centrifugation for 30 min at 15,000 rpm, the supernatant was subjected
to phenyl Sepharose column chromatography (5-mm inner diameter by 5 cm). The column was preequilibrated with buffer B (3 mM EDTA, 10 mM Tris-HCl, pH 7.4) containing 1 M
(NH4)2SO4. The fraction containing the ATPase activity was obtained by elution of buffer B with
a reverse linear (NH4)2SO4 gradient
(1 to 0 M).
Other methods.
N-terminal amino acid sequence analysis was
performed by using a Shimadzu PPSQ-10 protein sequencer equipped with a
Shimadzu C-R7A analyzer (Kyoto, Japan) after transferring a protein
from a sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) gel onto a nylon filter. Protein concentration was
determined by the bicinchronic acid method (29), using
bovine serum albumin as a standard (Pierce Chemical Company). The
ATPase activity was measured at 25°C in the presence of an
ATP-regenerating system (32). The putative genes were
originally identified as open reading frames (ORFs) by a software,
Genetyx-Mac version 9.0 (Software Development Co., Ltd.). Then, a
function was inferred from DNA homology matching. Homologies were
determined by utilizing the BLAST network service (2) from
National Center for Biotechnology Information.
Nucleotide sequence accession number.
The nucleotide
sequence data reported in this paper will appear in the DDBJ, EMBL, and
GenBank nucleotide sequence databases with the accession no.
AB022018.
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RESULTS |
Cloning and DNA sequencing of the genes of ORFs 1 to 5.
A region in the 17-kbp insert of MK27 genomic DNA was subcloned into
a pUC118 vector, and the nucleotide sequence was determined. Five ORFs
that were preceded by possible Shine-Dalgarno sequences were found in
it. However, no possible promoter and terminator regions could be
found. Therefore, these five ORFs seemed to be transcribed by the same
promoter, located upstream of this DNA fragment.
ORF-4 comprises 1,416 nucleotides, which begins with an ATG initiation
codon at nucleotide 3,078 from the 5' end of the region
shown in Fig.
1. The sequences similar
to the primers initially
designed for PCR were also in it. The derived
amino acid sequence
(included in Fig.
1 as well) composed of 471 amino
acid residues
is highly homologous with those of
F
1-ATPase
subunits (Table
2). Its size is also reasonable in
comparison with those from
other bacterial species. The crystal
structure of the F
1-ATPase
from the bovine
mitochondrion has shown that each of
and
subunits
consists of
three domains; N-terminal, central, and C-terminal
domains
(
1). The corresponding regions of the ORF-4 product
from
D. vulgaris Miyazaki F are the region of 1 to 82 (N-terminal
domain), 83 to 357 (central domain), and 358 to 471 (C-terminal
domain)
in terms of amino acid sequence. In the central domain,
the P loop,
which interacts with the phosphate region of a bound
nucleotide, and
the region around Glu-188 are highly homologous
with those of other
subunits. The side chain of Glu-188 is assumed
to catalyze ATP
hydrolysis (
1). Now, we can conclude that the
ORF-4 product
is the F
1-ATPase
subunit.
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FIG. 1.
Nucleotide sequences of the atpH,
atpA, atpG, atpD, and atpC
genes from D. vulgaris Miyazaki F and deduced amino acid
sequences of the five atp gene products. Nucleotides are
numbered from the 5' end of the 4,950-bp region presented. The putative
ribosome binding sites from the five atp genes are doubly
underlined. The P-loop amino acid sequence of the -subunit is
shaded.
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TABLE 2.
The amino acid sequence homologies between those encoded
by D. vulgaris Miyazaki F atp genes and those of
other bacteria
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ORF-2 comprises 1,467 nucleotides, which begins with an ATG initiation
codon at nucleotide 654 from the 5' end. The derived
amino acid
sequence (included in Fig.
1) composed of 488 amino
acid residues is
highly homologous with those of F
1-ATPase
subunits
(Table
2) and is a reasonable size compared with those from other
bacterial species. A sequence corresponding to the P loop
(G-D-R-Q-T-G-K-T
starting from nucleotide 1,158) is also present.
Consequently,
the ORF-2 product must be the
F
1-ATPase
subunit.
ORF-3 comprises 885 nucleotides, which begins with an ATG initiation
codon at nucleotide 2,176 from the 5' end. The derived
amino acid
sequence (included in Fig.
1) composed of 294 amino
acid residues shows
certain homology with those of F
1-ATPase
subunits
(Table
2). The relatively low homology is a general
feature among the
F
1-ATPase
subunits from various bacterial
species.
ORF-3 has little homology with other subunits of the
F
1-ATPases of other bacterial species. Furthermore, the
size of
the ORF-3 product is reasonable in comparison with
subunits
from other bacterial species. Therefore, the ORF-3 product should
be
the F
1-ATPase
subunit.
ORFs 1 and 5 comprise 519 and 405 nucleotides, respectively. The ORF-1
begins with a GTG initiation codon at 131, and the
derived amino acid
sequence (included in Fig.
1) composed of 172
amino acid residues is a
little homologous with those of F
1-ATPase
subunits
of various species (Table
2). ORF-5 begins with an
ATG initiation codon
at 4,504, and the derived amino acid sequence
(included in Fig.
1)
composed of 134 amino acid residues shows
certain homology with those
of F
1-ATPase
subunits of various
species.
Furthermore, ORF-1 and ORF-5 have little homology with
other subunits
of other bacteria. The sizes of these products
also are reasonable as
the
and
subunits, respectively. Now,
it can be concluded that
the ORF-1 and ORF-5 products are the
F
1-ATPase
and
subunits,
respectively.
The organization of the genes mentioned above is presented in Fig.
2. The
F
oF
1-ATPase genes are known to form a
cluster in
general (
22). The gene arrangement in the order
of the
,
,
,
, and
subunit genes in
D. vulgaris Miyazaki F is in accordance
with those found for other
species (
34). This also supports
the conclusion that
D. vulgaris Miyazaki F has an F-type ATPase
(or
ATP synthase). Since the operon of F-type ATPase genes usually
has
F
o genes upstream of the F
1 genes, it is
natural that the
analyzed fragment does not include a promoter region.
The GC contents
of codon usage for
atpH,
atpA,
atpG,
atpD, and
atpC genes were
62.2, 63.7, 63.1, 62.6, and 64.0%, respectively. The GC contents
of a total
of 4,950 bp sequenced in this study were 62.8%, in
contrast to 61.7%
for cytochrome
c3 (
16) and 74.4% for
flavin
mononucleotide-binding protein (
14), 66.2% for
[NiFe] hydrogenase
(
8), and 62.1% for cytochrome
c553 (
15) from
D. vulgaris Miyazaki F. Thus, the codon preference should be similar to that
of
other genes of
D. vulgaris Miyazaki F.
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FIG. 2.
The organization of atp genes of D. vulgaris Miyazaki F. ORFs are represented by boxes. The numbers in
the atpH to atpC boxes indicate those of the
amino acid residues. The horizontal arrows with numbers indicate the
fragments used in various subclones designated pMK1, pMK2, pMK3, and
pMK4, respectively (see Table 1). The black box of MK27 genomic DNA
indicates the cloned 17-kbp fragment.
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Purification of a component with ATPase activity from
D. vulgaris Miyazaki F.
A component with
ATPase activity that was purified from D. vulgaris
Miyazaki F membranes was described under Materials and Methods. As
shown in Table 3, the specific activity
increased approximately 100-fold during the three steps of
purification. SDS-PAGE of the fractions with ATPase activity showed
the presence of three major bands corresponding to the molecular masses
of 60.5, 51.5, and 34.7 kDa (Fig. 3). To
identify these proteins, each of these bands was transferred
electrophoretically from a SDS-12% PAGE gel onto a nylon filter, cut
separately, and analyzed by a protein sequencer. The sequence of the N
terminus of the largest one among the three bands was determined to be
Met-Gln-Ile-Lys-Ala-Glu-Glu-Ile-Ser-Lys-..., which is identical to
that of the expected product of the atpA gene. The
N-terminal sequence of the second largest one was
Ser-Ala-Asn-Ile-Gly-Lys-Ile-Val-Gln-Val-Ile-Gly-Ala-Val-Val-Asp-Val-Glu-Phe-Pro-..., which is identical to that of the expected product of the
atpD gene, with the initial methionine residue cleaved. The
sequence of the third one was
Pro-Ser-Leu-Lys-Asp-Val-Lys-Val-Lys-Ile-Ala-Gly-Val-Lys-Lys-Thr-Lys-Gln-Ile-Thr-Lys-Ala-Met-Asn-Met-Val-Ala-..., which is identical to that of the expected product of the
atpG gene, with the initial methionine residue cleaved.
Therefore, it can be concluded that the F1-ATPase genes
are actually expressed in D. vulgaris Miyazaki F cell
membranes under physiological conditions.
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FIG. 3.
SDS-12% PAGE analysis of the relevant fraction at each
purification step. The fractions with ATPase activity were analyzed
by SDS-12% PAGE and stained with Coomassie brilliant blue. The
molecular mass markers in lanes 1 and 7 are composed of phosphorylase
b (97,400 Da), bovine serum albumin (66,200 Da), ovalbumin
(45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor
(21,500 Da), and lysozyme (14,000 Da). Lane 2, the cytoplasmic membrane
fraction; lane 3, the supernatant after chloroform treatment; lane 4, after MonoQ elution; lane 5, after phenyl Sepharose elution; lane 6, subunit purified from thermophilic bacterium PS 3.
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DISCUSSION |
The whole genes of the , , , , and subunits of
F1-ATPase of a sulfate-reducing bacterium have been
cloned and sequenced for the first time in this work. Furthermore, the
actual presence of the gene products in the D. vulgaris
Miyazaki F membranes was also confirmed. Therefore, this should be
present as F1Fo-ATPase in the cell,
although its actual components and their stoichiometry are not yet
established. To understand the biological role of the ATPase gene
products in D. vulgaris Miyazaki F cells, its specific
activity was compared with that of E. coli membranes. E. coli JM109 was cultured aerobically overnight at 37°C.
The membrane fraction of the E. coli cells was prepared
in the same way as that of D. vulgaris Miyazaki F. The
specific ATPase activity of the membrane fraction of E. coli was 0.078 µmol/min/mg protein. This is similar to the
specific activity of the D. vulgaris Miyazaki F membrane
fraction shown in Table 3. Therefore, D. vulgaris Miyazaki F
should have a similar amount of F1Fo-ATPase
in the cytoplasmic membrane as E. coli does under aerobic
conditions. F1Fo-ATPase in E. coli works for energy production, namely for ATP synthesis under
aerobic conditions. The presence of a large amount of ATPase in the
D. vulgaris Miyazaki F cell strongly suggests that the gene
product is actually the F-type ATP synthase and is working to
synthesize ATP in the D. vulgaris Miyazaki F cell even under
anaerobic conditions. The amount is too much for the F1Fo-ATPase to work as a proton pump at
expense of ATP in the neutral pH region.
The sulfate-reducing bacteria contain abundant cytochromes
although they are strict anaerobes (20). These cytochromes
should be involved in electron transport systems in sulfate
respiration. Some sulfate-reducing bacteria can grow well by using
molecular hydrogen as a sole energy source (3). Furthermore,
an increase of the H+ concentration outside of the
cytoplasmic membrane coupled with hydrogen consumption and sulfite
reduction had been reported for D. vulgaris Miyazaki
(17). This observation indicates generation of the
H+ concentration gradient across the cytoplasmic membrane
by the electron transport from hydrogen to sulfite. Therefore, it can be concluded that the products of the F1-ATPase genes
are involved in ATP synthesis, utilizing the H+ gradient
generated by the electron transport system in the sulfate respiration.
This is the first evidence showing the presence of the F-type ATP
synthase associated with sulfate respiration. In 1981, Odom and Peck
proposed a chemiosmotic hydrogen cycling model for energy coupling in
Desulfovibrio species. This model has predicted that
Desulfovibrio can produce ATP by oxidative phosphorylation and should have a H+-ATP synthase. Our conclusion is
consistent with their model. However, the possibility for the presence
of proton pumps in the electron transport system cannot be removed. The
ATP synthesis coupled to nitrite respiration in Desulfovibrio
gigas membrane vesicles was reported in connection with generation
of the proton gradient (4). Our conclusion can explain this
observation as well.
The role of F-type H+-ATP synthase in strict anaerobes is
not yet well understood. It is inferred to work as an ATPase
coupled with proton pumping in some strict anaerobes. On the other
hand, there are some examples functioning as an ATP synthase. An
Na+-translocating F1Fo-ATPase
in P. modestum is known to synthesize ATP, utilizing the
Na+ gradient across the cytoplasmic membrane
(11). A decarboxylation reaction is directly coupled with
Na+ pumping in the methylmalonyl-CoA decarboxylase. The
fermentation of succinate to propionate and CO2 operates at
a total free energy change of only 
20 kJ/mol (10). This
amount of energy is not sufficient to synthesize 1 mol of ATP from ADP
and inorganic phosphate. The free energy expense for ATP synthesis
under in vivo conditions is about 70 to 80 kJ/mol (31).
Thus, three or four decarboxylation reactions have to couple
with the synthesis of one ATP molecule. This was indicated as the major
reason for the presence of ATP synthase in P. modestum
(11). A H+-translocating
F1Fo-ATPase in M. thermoacetica
was also reported to synthesize ATP, utilizing the H+
gradient generated by an electron transport chain which contains a
menaquinone and two b-type cytochromes (13). The
detailed mechanism and role of this energy conversion are not yet
clear. It is also reported that the structure of the Fo
domain of M. thermoautotrophica ATPase is different from
that of the common F-type ATP synthase (5, 6).
In the case of sulfate-reducing bacteria, the oxidation of lactate to
acetate results in the generation of 1 mol of ATP and 4 mol of
electrons (or 2 mol of hydrogen molecules) at the substrate level. The
ATP synthesized at the substrate level is used to activate the sulfate
ion for reduction (20). The electrons are also used to
reduce sulfate ion. The total reaction can be written as follows (26): CH3CH(OH)COOH + (1/2)SO42 = CH3COOH + CO2 +H2O + (1/2)S2 
G° = 94
kJ
Since the free energy released from this
oxidation-reduction reaction is much larger than that of P. modestum, the energy conversion system in D. vulgaris
Miyazaki F should be different from that in P. modestum. The
free energy change of 94 kJ/mol is converted to ATP through electron
transport coupled with the H+ gradient generation. The
generation of H+ gradient is not directly coupled
with the oxidation of organic compounds, in contrast to P. modestum. This process is a prototype of oxygen respiration.
There would be three types of current strict anaerobes in terms of the
physiological role of F1Fo-ATPase. The most
primitive types use F1Fo-ATPase as an ATP
synthase without involvement of electron transport systems. The proton
or Na+ gradient is used for efficient accumulation of small
energy. The second type does not heavily rely on
F1Fo-ATP synthase for the energy production.
ATP synthesis at the substrate level in the fermentation should be the
major source of the energy in these anaerobes. In this case,
F1Fo-ATPase must be mainly used for the regulation of the cytoplasmic pH. The third type of strict anaerobes heavily relies on F1Fo-ATP synthase in
association with electron transport systems for the energy production.
The energy conversion system in the third type of strict anaerobes can
be placed between the fermentation and oxygen respiration from the
evolutional point of view. The sulfate-reducing bacteria are one of the
major groups of the third type.
| |
ACKNOWLEDGMENTS |
We thank Kaeko Tozawa, Tokushima University, Keneath H. Nealson
of Jet Propulsion Laboratory at California Institute of Technology, and
Hideki Taguchi and Eiro Muneyuki at Tokyo Institute of Technology for
valuable discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan. Phone:
81-45-339-4232. Fax: 81-45-339-4251. E-mail:
akutsu{at}ynu.ac.jp.
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