JOURNAL OF BACTERIOLOGY, July 1998, p. 3448–3452
0021-9193/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 180, No. 13
The A1Ao ATPase from Methanosarcina mazei: Cloning of the
59 End of the aha Operon Encoding the Membrane Domain
and Expression of the Proteolipid in a Membrane-Bound
Form in Escherichia coli
CLAUDIA RUPPERT, SÖNKE WIMMERS, THORSTEN LEMKER,
AND
VOLKER MÜLLER*
Lehrstuhl für Mikrobiologie, Ludwig-Maximilians-Universität München, 80638 München, Germany
Received 5 January 1998/Accepted 16 April 1998
tial membrane-spanning subunits were found in that study
(35). In order to determine unequivocally the subunit composition of the A1Ao ATPase from the methylotrophic M. mazei,
we sequenced the region upstream of ahaE and found three
more ATPase genes. Two encode subunits of the membrane
domain, the proteolipid and the homolog of the 100-kDa
polypeptide from V1Vo ATPases. The third encodes a hydrophilic peptide similar to deduced gene products of previously
unassigned function in M. thermoautotrophicum and M. jannaschii.
We will show here that the proteolipid of M. mazei is expressed in Escherichia coli and is targeted to the cytoplasmic
membrane, which will facilitate future work on the analysis of
the structure and function of the proton conductance pathway
in methanoarchaeal ATPases.
A major goal in understanding the physiology of methanogens has been to delineate the mechanism by which they produce ATP. In recent years it has become evident that methanogenic archaea do not synthesize ATP by substrate level
phosphorylation but couple the conversion of all substrates
known to the generation of ion gradients, H1 and Na1, across
the cytoplasmic membrane (5, 19). All methanogens investigated so far contain a H1-translocating A1Ao ATPase. These
enzymes have been purified from a number of organisms, but
depending on the isolation procedure, two to six nonidentical
subunits have been found (3, 11–14, 26, 35). Recently, the
genome sequencing projects revealed eight putative ATPase
genes on the chromosomes of Methanobacterium thermoautotrophicum and Methanococcus jannaschii, as deduced from
sequence comparisons (2, 28). However, the exact subunit
composition of the ATPase remains to be established.
In an ongoing project dealing with the elucidation of the
mechanism of ATP synthesis in methanogens, we have purified
the ATP synthase from Methanosarcina mazei and concluded
from biochemical and molecular data that the hydrophilic domain contains at least seven subunits and that the corresponding genes (ahaECFABDG) are clustered on the chromosome
(35). Furthermore, a 36- and a 7-kDa polypeptide were found
in the purified enzyme and were assigned to the membrane
domain; the 7-kDa polypeptide was identified as the proteolipid. Unfortunately, neither the genes encoding the 36- and
7-kDa polypeptides nor any genes encoding any other poten-
MATERIALS AND METHODS
Organisms and plasmids. M. mazei Gö1 (DSM 3647) was obtained from the
Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig,
Federal Republic of Germany. M. mazei was grown under strictly anaerobic
conditions as described by Hippe et al. (9). Wild-type Saccharomyces cerevisiae
(SF838-1Då MATa ade6 leu2-3 leu2-112 ura3-52 his4-519 pep4-3 gal2) and
vma3D (SF838-1Då vma3-D1, isogenic with SF838-1Då except for vma3D::ura),
vma11D (RHA107, isogenic with SF838-1Då except for vma11D::leu), and
vma16D (LGY10, isogenic with SF838-1Då except for vma16D::leu) mutants
were obtained from T. H. Stevens, University of Oregon, Eugene, and were
grown on synthetic dropout medium on yeast extract-peptone-dextrose (YEPD)
at pH 5.0 or 7.0 as described elsewhere (27, 36). E. coli DH5a (supE44 DlacU169
F80 lacZDM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 [8]), DK8 (1100D[uncBuncC] ilv::Tn10 [16]), MJM413 (F1 asnA1 asnB31 thi-1 recA56 srl-1300::Tn10
atpE1003 [leu-43amber] [7]), and LE392 (supE44 supF58 hsdR514 galK2 galT22
metB trpR55 lacY1 [22]) were grown at 37°C on Luria-Bertani medium or on
minimal medium supplemented with methionine and tryptophan (each at 50
mg/ml). Where indicated, either 1% glucose or 1% succinate was used as a
carbon and energy source (31). Plasmids used were pJLA603 (25), pHSG398,
* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Ludwig-Maximilians-Universität München, Maria-Ward-Str. 1a,
80638 München, Germany. Phone: 49-8917919836. Fax: 49-8917919855.
E-mail: v.mueller@lrz.uni-muenchen.de.
3448
Downloaded from jb.asm.org at Molecular Pharmacology, Medical School Hannover on April 25, 2008
Three additional ATPase genes, clustered in the order ahaH, ahaI, and ahaK, were found upstream of the
previously characterized genes ahaECFABDG coding for the archaeal A1Ao ATPase from Methanosarcina mazei.
ahaH, the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis
revealed that the clusters ahaHIK and ahaECFABDG are transcribed as one message. AhaH is a hydrophilic
polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated
ATPase genes in Methanobacterium thermoautotrophicum and Methanococcus jannaschii. AhaI has a two-domain
structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane
a helices. It is similar to the 100-kDa polypeptide of V1Vo ATPases and is therefore suggested to participate
in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane a helices and, on
the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide
which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from M. thermoautotrophicum and M. jannaschii, respectively. ahaK is expressed in Escherichia coli, and
it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea
and bacteria. This is the first report on the expression and incorporation into E. coli lipids of a membrane
integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the
membrane domain of the methanoarchaeal ATPase.
A1Ao ATPase FROM METHANOSARCINA MAZEI
VOL. 180, 1998
FIG. 1. DNA and deduced amino acid sequences of the 59 terminus of the
aha operon. The start of ahaH is indicated. Conserved residues of putative
promoter sequences (boxes A and B) are marked by asterisks. The putative
ribosome binding site of ahaH is underlined.
A1Ao ATPase, since the ahaHIK cluster is separated from the
larger cluster by 211 bp and since M. mazei has been shown to
possess two structurally related ATPases (1). When total RNA
was isolated from cells grown on methanol and was hybridized
against ahaE or ahaK, the same pattern was observed, indicating that both clusters belong to the same transcriptional unit
(Fig. 2). The 9.0-kb transcript corresponds well to a mRNA
covering ahaH through ahaG. Whether the 6.8-kb transcript is
derived from a second transcriptional start point or by a modification of the 9.0-kb transcript remains to be elucidated. The
0.64-kb transcript hybridizing to the proteolipid-encoding
gene, ahaK, indicates an additional definite transcription of
ahaK only. This is of particular importance because the proteolipid is present in 9 to 12 copies per ATPase molecule. In
bacteria, enhanced synthesis of the proteolipid is achieved by
translational but not by transcriptional regulation (18). Putative, but not well-conserved, promoter sequences are found
approximately 0.6 kb upstream of a weak transcriptional terminator downstream of ahaK.
AhaH. AhaH has a molecular weight of 12,200, consists of
109 residues, and has a deduced pI of 4.74. The peptide is
highly charged; 21 and 16% of the residues are acidic and
basic, respectively. Database searches identified the following
deduced gene products with previously unassigned function as
homologs of AhaH: MJ0223 of M. jannaschii (25% identity)
(2), MT0961 of M. thermoautotrophicum (25% identity) (28),
and AF1158 of Archaeoglobus fulgidus (29% identity) (15).
RESULTS
Nucleotide sequence of the 5* end of the aha operon from M.
mazei and its transcriptional analysis. In a previous study, the
genes ahaECFABDG were found to encode hydrophilic subunits of the A1Ao ATPase of M. mazei (35). The DNA sequence upstream of ahaE of M. mazei was determined. Directly upstream of ahaE is an apparently noncoding, AT-rich
region of 211 bp. Upstream of the intergenic region, three
ATPase genes, designated ahaH, ahaI and ahaK, were identified; they are 330, 1,950, and 243 bp long, respectively. ahaH
overlaps with ahaI by 8 bp, and ahaI and ahaK are separated by
only 4 bp. Each of the genes is preceded by a well-placed and
well-conserved Shine-Dalgarno sequence, and translation is
initiated by an ATG codon in all cases. Upstream of ahaH is an
AT-rich region which contains two potential archaeal promoter sequences (Fig. 1). The DNA sequence (1,200 bp) upstream of this potential promoter sequence does not contain
any ATPase genes.
The gene cluster ahaECFABDG clearly codes for subunits of
the A1Ao ATPase (35). However, sequence comparisons alone
do not allow the assignment of AhaHIK to subunits of the
FIG. 2. Northern blot of total RNA of methanol-grown cells of M. mazei.
Lane 1, autoradiograph of a sample probed with ahaK; lane 2, autoradiograph of
a sample probed with ahaE; lane 3, total RNA in formaldehyde agarose gel; lane
4, RNA standard in formaldehyde agarose gel.
Downloaded from jb.asm.org at Molecular Pharmacology, Medical School Hannover on April 25, 2008
pHSG399 (30), pVT100-U, pVT102-U (32), and pRT1001, which is an XbaI
subclone of pEW1 (35).
For the expression studies, ahaK was amplified by PCR by introducing an NdeI
restriction site at the 59 end and a BamHI restriction site at the 39 end. The
252-bp fragment containing ahaK of M. mazei (primer ahaK/U [59-GAATAAA
CATATGGTAGACGCAGCA-39] and primer ahaK/R [59-TAGGATCCTAAA
TTTAAGTAAAATC-39]) was cloned into pJLA603, giving rise to pRT200. To
complement the proteolipid mutant of E. coli, MJM413, plasmids pRT201
(pHSG398::ahaK) and pRT202 (pHSG399::ahaK) were constructed by cloning
the XhoI/BamHI fragment of pRT200 into pHSG399 and pHSG398; in pRT202
and pRT201, ahaK is oriented colinear and with opposite polarity to the lac
promoter, respectively. For complementation studies of the Dvma11::leu and
Dvma16::leu proteolipid mutants of S. cerevisiae, the ahaK gene was cloned into
the yeast expression vectors pVT100-U and pVT102-U under the control of the
adh promoter. To complement the S. cerevisiae, Dvma3::ura mutant, a leucine
cassette was inserted into the uracil cassette in pVT100-U and pVT102-U, giving
rise to pVM1001 and pVM1002, respectively. The ahaK gene was cloned into the
four above-mentioned plasmids after digestion of pRT201 with PstI and BamHI.
The resulting plasmids were named pRT203 (pVT100-U::ahaK), pRT204
(pVT102-U::ahaK), pVM1003 (pVM1001::ahaK), and pVM1004 (pVM1002::
ahaK). In pRT203 and pVM1003, the gene is oriented 59339 with respect to the
adh promoter; in pRT204 and pVM1004, it is oriented with the opposite polarity.
Northern blotting. RNA was prepared from M. mazei grown on methanol (200
mM). Cells were harvested anaerobically (4,000 rpm; 10 min; 4°C) at an optical
density at 600 nm (OD600) of 0.6, and RNA isolation and blotting were carried
out as described elsewhere (22), with the following modifications. The pellet was
resuspended in 30 mM sodium acetate (pH 5.5)–1.5% sodium dodecyl sulfate)
(SDS), and RNA was isolated by extraction with phenol (equilibrated with 20
mM sodium acetate–1 mM EDTA–0.1% [wt/vol] SDS [pH 5.5]) and chloroformisoamyl alcohol (24:1) at 65°C.
Molecular procedures. All procedures were performed according to standard
techniques (22). DNA sequences were determined from nested deletion clones
by the chain termination method of Sanger et al. and were analyzed on a UNIX
computer by using the Genetics Computer Group package (6, 23).
Expression studies. For complementation assays, E. coli MJM413 was transformed with the plasmids indicated and plated on Tanaka plates containing
glucose and chloramphenicol. Transformants were streaked in parallel on
Tanaka plates containing glucose and chloramphenicol and on Tanaka plates
containing succinate and chloramphenicol in the absence or presence of isopropyl-b-D-thiogalactopyranoside (IPTG) and were incubated at different temperatures. Yeast competent cells were transformed with the plasmids indicated and
plated on synthetic dropout medium. Transformants were streaked in parallel on
YEPD plates at pH 5.0 or 7.0 and were incubated at 30°C. For heterologous
expression of AhaK in E. coli DK8, competent cells were transformed with the
plasmids indicated, and expression was achieved by thermal induction (42°C)
after the cultures reached an OD600 of 0.1. The cultures remained at 42°C for
another 3 to 4 h. SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 12% polyacrylamide gels according to the procedure given in reference 24.
Immunological studies. Western blotting with SDS-polyacrylamide gels was
performed as described elsewhere (35). Polyclonal antibodies against AhaK (34)
were obtained from F. Mayer, Göttingen, Federal Republic of Germany.
Nucleotide sequence accession number. The nucleotide sequences reported in
this paper have been submitted to GenBank under accession no. U47274.
3449
3450
RUPPERT ET AL.
Twenty-three and 44% of the amino acids of AhaH are identical and similar, respectively, to those of NtpF of Enterococcus
hirae (29). Therefore, there is compelling reason to designate
MJ0223, MT0961, and AF1158 atpHMj, atpHMt, and atpHAf.
The fact that ahaH, atpHMj, atpHMt, and atpHAf are the first
genes in their operons suggests that they could correspond to
uncI of bacterial F1Fo ATPases (33), but only 12% of the
amino acids of UncI of E. coli and AhaH were found to be
identical.
AhaI. ahaI encodes a peptide of 649 residues with a molecular weight of 72,048 and a calculated pI of 5.80. Twenty-seven,
30, and 35% of its residues are conserved in AtpIMt, AtpIMj,
and AtpIAf, respectively, and 20 (Vph1p of S. cerevisiae [17]) to
24% (Vph1p of Bos taurus [21]) of its residues are conserved in
the 100-kDa subunits of the V1Vo ATPases of eucarya. Twenty-seven percent of the residues of NtpI from E. hirae are
identical to those of AhaI. Hydrophobicity plots of AhaI pro-
pose a highly hydrophilic N-terminal domain of 39 kDa and a
hydrophobic C-terminal domain of 33 kDa. Garnier analysis of
the hydrophilic domain of AhaI predicts a highly a-helical
structure, as is the case with subunit b of the F1Fo ATPases.
Interestingly, the similarities of the hydrophilic domain of subunit b of the F1Fo ATPases to the hydrophilic domains of AhaI
and AtpIMj are 22 and 26%, respectively. The hydrophobic C
termini of AhaI and AtpI are predicted to have six to seven
putative transmembrane helices. The similarity to subunit a of
the F1Fo ATPases is below 20%.
AhaK. ahaK codes for a protein with 80 residues and a
molecular mass of 7.9 kDa. The pI was determined to be 4.04.
Hydrophobicity plots predict one hairpin with two transmembrane a helices. It is very similar to proteolipids from other
archaea such as M. thermoautotrophicum (35% identity) (28),
M. jannaschii (45% identity) (2), A. fulgidus (52% identity)
(15), Sulfolobus acidocaldarius (31% identity) (4), and Halobacterium salinarum (52% identity) (10). Thirty-three percent
of the residues are conserved in AhaK and NtpK from E. hirae
(29). On the basis of its molecular mass, AhaK is more similar
to the proteolipids of bacteria, but on the basis of sequence
analysis, it is more closely related to the proteolipids of V1Vo
ATPases from eucarya; the degrees of identity range from 26.7
to 31.4%. The active carboxylate of helix 2, which is conserved
in all proteolipids known so far, is present at position 65 (Fig.
3). Since the proteolipid is essential for proton translocation, it
was analyzed in more detail.
Expression of the proteolipid in E. coli. To facilitate a biochemical and biophysical analysis of the proteolipid from
methanogens, we cloned ahaK from M. mazei into the expression vector pJLA603, and the resulting plasmid, pRT200, was
transformed into the ATPase-negative E. coli strain DK8.
Upon induction of expression, the growth of the host cells
ceased (data not shown). Cells were harvested and separated
into cytoplasmic and membrane fractions and were subjected
to SDS-PAGE. Only the membranes of E. coli DK8(pRT200)
contained a polypeptide of 7 kDa, which cross-reacted with an
antibody directed against the proteolipid of M. mazei (Fig. 4).
Interestingly, the proteolipid was inserted into the E. coli cytoplasmic membrane despite the different chemical natures of
lipids from bacteria and archaea. This offers a new opportunity
FIG. 4. SDS-PAGE and Western blot of the proteolipid from M. mazei expressed in E. coli. ahaK was expressed by raising the temperature to 42°C at an OD600
of 0.1. Cells were harvested at an OD600 of 1. Protoplasts were prepared, lysed by osmotic shock, and separated into cytoplasm and membrane fractions, and the
membrane fractions were subjected to SDS-PAGE (a) and Western blotting (b). Lanes 1, DK8(pJLA603), not induced; lanes 2, DK8(pJLA603), induced; lanes 3,
DK8(pRT200), not induced; lanes 4, DK8(pRT200), induced.
Downloaded from jb.asm.org at Molecular Pharmacology, Medical School Hannover on April 25, 2008
FIG. 3. Alignment of the proteolipids from M. mazei (Mma), M. thermoautotrophicum (Mth) (28), M. jannaschii (Mja) (2), A. fulgidus (Afu) (15), H.
salinarum (Hsa) (10), and S. acidocaldarius (Sac) (4). Conserved residues are
shaded, and putative transmembrane segments are boxed. The boundaries of the
transmembrane segments are hypothetical. Leader sequences of the proteolipids
from H. salinarum and S. acidocaldarius are not shown.
J. BACTERIOL.
A1Ao ATPase FROM METHANOSARCINA MAZEI
VOL. 180, 1998
3451
to study the proteolipid from methanogens in a heterologous
system.
DISCUSSION
The A1Ao ATPase operon from M. mazei and its implications
for the subunit composition of methanoarchaeal ATPases. The
molecular data revealed that the A1Ao ATPase of M. mazei is
encoded by 10 genes, ahaH through ahaG (Fig. 5). Apparently,
the gene structure at the 59 end is identical in all archaeal A1Ao
and bacterial V1Vo ATPase operons known so far. It is noteworthy that AhaH and AhaI have homologs not in F1Fo
ATPases but in V1Vo ATPases. According to its molecular
mass, AhaK, the proteolipid, is similar to F1Fo ATPases, but its
primary structure is more similar to that of the proteolipids of
V1Vo ATPases than to those of F1Fo ATPases. Attempts to
genetically complement proteolipid mutants of E. coli and S.
cerevisiae failed (see Materials and Methods), although ahaK
was expressed in both, as shown by Western blots (data for
expression in S. cerevisiae are not shown). Apparently, it was
not assembled functionally into F1Fo or V1Vo ATPases. Taken
together, the subunit composition and any given polypeptide of
the methanoarchaeal A1Ao ATPase are more similar to V1Vo
than to F1Fo ATPase subunits. This demonstrates a close evolutionary linkage of A1Ao and V1Vo ATPases.
Structure of the membrane domain. From the data presented in this study, it is obvious that the Ao domain consists of
only two different subunits, the homolog of the 100-kDa subunit of V1Vo ATPases and the proteolipid. This is in contrast
to previous results in which a 36-kDa protein, but not AhaI,
was found in the membrane fraction of the purified enzyme
(35). However, it is conceivable that AhaI is sensitive to proteolysis, and a loss of the hydrophilic domain during the purification procedure would leave only a ;36-kDa domain in the
membrane, as reported for the 100-kDa subunits of V1Vo
ATPases.
One of the major differences between A1Ao and V1Vo
ATPases known hitherto was the sizes of their proteolipids;
this obvious difference was believed to be the reason for the
apparent inability of V1Vo ATPases to synthesize ATP (20).
An experimental approach verifying or questioning this assumption was hindered by the fact that the 7-kDa proteolipid
from M. mazei was not assembled functionally into the F1Fo
ATPase from E. coli or the V1Vo ATPase from S. cerevisiae
(see above). Fortunately, a solution to this interesting question
comes with the genomic sequences from M. jannaschii and M.
thermoautotrophicum (2, 28). The proteolipid of M. thermoautotrophicum is predicted to be 15.6 kDa with four transmem-
brane helices, and that of M. jannaschii is predicted to be 21.3
kDa with six transmembrane helices. From the genomic sequences it is evident that the A1Ao ATPases are the only
ATPases present in M. jannaschii and M. thermoautotrophicum.
Since these organisms gain energy only by ion gradient-driven
phosphorylation, there is no doubt that they function as ATP
synthases, and direct experimental proof that the A1Ao ATPases from methanogens function as ATP synthases is available (19).
Two important conclusions have to be drawn from these
observations: first, duplication of the proteolipid genes is already observed in archaea, and second, it not accompanied by
a failure of the ATPase to synthesize ATP. Therefore, the
apparent inability of V1Vo ATPases to synthesize ATP can no
longer be attributed to the size of the proteolipid respective to
its number of transmembrane a helices. More likely to be
important is the number of active carboxylates per ATPase.
Assuming a constant number of 12 hairpins per enzyme molecule, the F1Fo ATPase contains 12 glutamates, the A1Ao
ATPases of M. thermoautotrophicum and M. mazei also contain
12, and the A1Ao ATPase of M. jannaschii contains 8, but the
V1Vo ATPases contain only 6.
ACKNOWLEDGMENTS
This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
We are grateful to F. Mayer, University of Göttingen, for the generous gift of the AhaK-specific antibody. V.M. appreciated the warm
welcome, stimulating discussions, and introduction to yeast genetics by
T. H. Stevens, L. A. Graham, and colleagues during a stay in Eugene,
Oreg., in which the expression studies of ahaK in yeast were initiated.
REFERENCES
1. Becher, B., and V. Müller. 1994. DmNa1 drives the synthesis of ATP via an
DmNa12 translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1. J. Bacteriol. 176:2543–2550.
2. Bult, C. J., O. White, G. J. Olsen, L. X. Zhou, R. D. Fleischmann, G. G.
Sutton, J. A. Blake, L. M. Fitzgerald, R. A. Clayton, J. D. Gocaynee, A. R.
Kerlavage, B. A. Dougherty, J. F. Tomb, M. D. Adams, C. I. Reich, R.
Overbeek, E. F. Kirkness, K. G. Weinstock, J. M. Merrick, A. Glodek, J. L.
Scott, N. S. M. Geoghagen, J. F. Weidman, J. L. Fuhrmann, D. Nguyen, T. R.
Utterback, J. M. Kelley, J. D. Peterson, P. W. Sadow, M. C. Hanna, M. D.
Cotton, K. M. Roberts, M. A. Hurst, B. P. Kaine, M. Borodovsky, H.-P.
Klenk, C. M. Fraser, H. O. Smith, C. R. Woese, and J. G. Venter. 1996.
Complete genome sequence of the methanogenic archaeon, Methanococcus
jannaschii. Science 273:1058–1073.
3. Chen, W., and J. Konisky. 1993. Characterization of a membrane-associated
ATPase from Methanococcus voltae, a methanogenic member of the Archaea. J. Bacteriol. 175:5677–5682.
4. Denda, K., J. Konishi, T. Oshima, T. Date, and M. Yoshida. 1989. A gene
encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex. J. Biol. Chem. 264:7119–7121.
Downloaded from jb.asm.org at Molecular Pharmacology, Medical School Hannover on April 25, 2008
FIG. 5. Organization of genes in methanoarchaeal A1Ao ATPase operons. Genes encoding hydrophobic subunits are marked by asterisks. Homologous genes are
depicted by the same pattern. Data are from references 2 and 28.
3452
RUPPERT ET AL.
20. Nelson, N. 1992. Evolution of organellar proton-ATPases. Biochim. Biophys.
Acta 1100:109–124.
21. Peng, S. B., B. P. Crider, X.-S. Xie, and D. K. Stone. 1994. Alternative
mRNA splicing generates tissue-specific isoforms of 116-kDa polypeptide of
vacuolar proton pump. J. Biol. Chem. 269:17262–17266.
22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
23. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.
24. Schägger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range
from 1 to 100 kDa. Anal. Biochem. 166:369–379.
25. Schauder, B., H. Blöcker, R. Frank, and J. E. G. McCarthy. 1987. Inducible
expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene 52:279–283.
26. Scheel, E., and G. Schäfer. 1990. Chemiosmotic energy conservation and the
membrane ATPase of Methanolobus tindarius. Eur. J. Biochem. 187:727–735.
27. Sherman, F., G. R. Fink, and J. B. Hicks. 1982. Methods in yeast genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
28. Smith, D. R., L. A. Doucette-Stamm, C. Deloughery, H. Lee, J. Dubois, T.
Aldredge, R. Bashirzadeh, D. Blakely, R. Cook, K. Gilbert, D. Harrison, L.
Hoang, P. Keagle, W. Lumm, B. Pothier, D. Qiu, R. Spadafora, R. Vicaire,
Y. Wang, J. Wierzbowski, R. Gibson, N. Jiwani, A. Caruso, D. Bush, H. Safer,
D. Patwell, S. Prabhakar, S. McDougall, G. Shimer, A. Goyal, S. Pietrokovski, G. M. Church, C. J. Daniels, J.-I. Mao, P. Rice, J. Nölling, and J. N.
Reeve. 1997. Complete genome sequence of Methanobacterium thermoautotrophicum DH: functional analysis and comparative genomics. J. Bacteriol.
179:7135–7155.
29. Takase, K., S. Kakinuma, I. Yamato, K. Konishi, K. Igarashi, and Y. Kakinuma. 1994. Sequencing and characterization of the ntp gene cluster for
vacuolar-type Na1-translocating ATPase of Enterococcus hirae. J. Biol.
Chem. 269:11037–11044.
30. Takeshita, S., M. Sato, M. Toba, W. Masahashi, and T. Hashimoto-Gotoh.
1987. High-copy-number and low-copy-number plasmid vectors for lacZacomplementation and chloramphenicol-resistance selection. Gene 61:63–74.
31. Tanaka, S., S. A. Lerner, and E. C. C. Lin. 1967. Replacement of a phosphoenolpyruvate-dependent phosphotransferase by a nicotinamide adenine
dinucleotide-linked dehydrogenase for the utilization of mannitol. J. Bacteriol. 93:642–648.
32. Vernet, T., D. Dignard, and D. Y. Thomas. 1987. A family of yeast expression
vectors containing the phage f1 intergenic region. Gene 52:225–233.
33. Walker, J. E., M. Saraste, and N. J. Gay. 1984. The unc operon. Nucleotide
sequence, regulation and structure of ATP-synthase. Biochim. Biophys. Acta
768:164–200.
34. Wilms, R. 1992. Ph.D. thesis. University of Göttingen, Göttingen, Germany.
35. Wilms, R., C. Freiberg, E. Wegerle, I. Meier, F. Mayer, and V. Müller. 1996.
Subunit structure and organization of the genes of the A1Ao ATPase from
the archaeon Methanosarcina mazei Gö1. J. Biol. Chem. 271:18843–18852.
36. Yamashiro, C. T., P. M. Kane, D. F. Wolczyk, R. A. Preston, and T. H.
Stevens. 1990. Role of vacuolar acidification in protein sorting and zymogen
activation: a genetic analysis of the yeast vacuolar proton-translocating
ATPase. Mol. Cell. Biol. 10:3737–3749.
Downloaded from jb.asm.org at Molecular Pharmacology, Medical School Hannover on April 25, 2008
5. Deppenmeier, U., V. Müller, and G. Gottschalk. 1996. Pathways of energy
conservation in methanogenic Archaea. Arch. Microbiol. 165:149–163.
6. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of
sequence analysis program for the VAX. Nucleic Acids Res. 12:387–395.
7. Fraga, D., and R. H. Fillingame. 1991. Essential residues in the polar loop
region of subunit c of Escherichia coli F1F0 ATP synthase definded by
random oligonucleotide-primed mutagenesis. J. Bacteriol. 173:2639–2643.
8. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557–580.
9. Hippe, H., D. Caspari, K. Fiebig, and G. Gottschalk. 1979. Utilization of
trimethylamine and other methyl compounds and methane formation by
Methanosarcina barkeri. Proc. Natl. Acad. Sci. USA 76:494–498.
10. Ihara, K., S. Watanabe, K. Sugimura, and Y. Mukohata. 1997. Identification
of proteolipid from an extremely halophilic archaeon, Halobacterium salinarum, as an N,N9-dicyclohexyl-carbodiimide binding subunit of ATP synthase.
Arch. Biochem. Biophys. 341:267–272.
11. Inatomi, K. 1996. ATP-dependent H1-pump activity in inverted vesicles of
Methanosarcina mazei Gö1 and characterization of membrane ATPase. J.
Bacteriol. 178:2424–2426.
12. Inatomi, K. I. 1986. Characterization and purification of the membranebound ATPase of the archaebacterium Methanosarcina barkeri. J. Bacteriol.
167:837–841.
13. Inatomi, K. I., Y. Kamagata, and K. Nakamura. 1993. Membrane ATPase
from the aceticlastic methanogen Methanothrix thermophila. J. Bacteriol.
175:80–84.
14. Inatomi, K. I., M. Maeda, and M. Futai. 1989. Dicyclohexylcarbodiimidebinding protein is a subunit of the Methanosarcina barkeri ATPase complex.
Biochem. Biophys. Res. Commun. 162:1585–1590.
15. Klenk, H.-P., R. A. Clayton, J.-F. Tomb, O. White, K. E. Nelson, K. A.
Ketchum, R. J. Dodsen, M. Gwinn, E. K. Hickey, J. D. Peterson, D. L.
Richardson, A. R. Kerlavage, D. E. Graham, N. C. Kyrpides, R. D. Fleischmann, J. Quackenbush, N. H. Lee, G. G. Sutton, S. Gill, E. F. Kirkness, B. A.
Dougherty, K. McKenny, M. D. Adams, B. Loftus, S. Peterson, C. I. Reich,
L. K. McNeil, J. H. Badger, A. Glodek, L. Zhou, R. Overbeek, J. D. Gocayne,
J. F. Weidman, L. McDonald, T. Utterback, M. D. Cotton, T. Spriggs, P.
Artiach, B. P. Kaine, S. M. Sykes, P. W. Sadow, K. P. D’Andrea, C. Bowman,
C. Fujii, S. A. Garland, T. M. Mason, G. J. Olsen, C. M. Fraser, H. O. Smith,
C. R. Woese, and J. C. Venter. 1997. The complete genome sequence of the
hyperthermophilic, sulphate-reducing archaeon Archaeglobus fulgidus. Nature 390:364–370.
16. Klionsky, D. J., W. S. A. Brusilow, and R. D. Simoni. 1984. In vivo evidence
for the role of the ε subunit as an inhibitor of the proton-translocating
ATPase of Escherichia coli. J. Bacteriol. 160:1055–1060.
17. Manolson, M. F., D. Proteau, R. A. Preston, A. Stenbit, B. T. Roberts, M. A.
Hoyt, D. Preuss, J. Mulholland, D. Botstein, and E. W. Jones. 1992. The
VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in
vivo assembly and activity of the yeast vacuolar H1-ATPase. J. Biol. Chem.
267:14294–14303.
18. McCarthy, J. E. G., H. U. Schairer, and W. Sebald. 1985. Translational
initiation frequency of atp genes from Escherichia coli: identification of an
intercistronic sequence that enhances translation. EMBO J. 4:519–526.
19. Müller, V., M. Blaut, and G. Gottschalk. 1993. Bioenergetics of methanogenesis, p. 360–406. In J. G. Ferry (ed.), Methanogenesis. Chapman & Hall,
New York, N.Y.
J. BACTERIOL.