Lehrstuhl für Mikrobiologie,
Ludwig-Maximilians-Universität München, 80638 München, Germany
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INTRODUCTION |
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, H+ and
Na+, across the cytoplasmic membrane (5, 19).
All methanogens investigated so far contain a
H+-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 potential 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.
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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 MAT
ade6 leu2-3 leu2-112
ura3-52 his4-519 pep4-3 gal2) and vma3
(SF838-1Då vma3-1,
isogenic with SF838-1Då except for
vma3::ura), vma11 (RHA107,
isogenic with SF838-1Då except for
vma11::leu), and vma16 (LGY10,
isogenic with SF838-1Då except for
vma16::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 DH5 (supE44 lacU169 
80
lacZM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1
[8]), DK8 (1100[uncB-uncC]
ilv::Tn10 [16]),
MJM413 (F+ asnA+ asnB31 thi-1 recA56
srl-1300::Tn10 atpE1003
[leu-4
amber] [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 µg/ml). Where indicated, either 1% glucose or 1% succinate was
used as a carbon and energy source (31). Plasmids used were pJLA603 (25), pHSG398, 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 5' end and a
BamHI restriction site at the 3' end. The 252-bp fragment
containing ahaK of M. mazei (primer ahaK/U
[5'-GAATAAACATATGGTAGACGCAGCA-3'] and primer ahaK/R
[5'-TAGGATCCTAAATTTAAGTAAAATC-3']) 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
vma11::leu and
vma16::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, vma3::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 5'3' 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 chloroform-isoamyl 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-
-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.
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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.
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Fig. 1.
DNA and deduced amino acid sequences of the 5' 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.
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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 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.
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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.
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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).
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 propose 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 -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 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.
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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.
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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 to
study the proteolipid from methanogens in a heterologous system.
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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.
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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
5' 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.
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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.
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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 transmembrane 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 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.
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.
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Abstract
Introduction
translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.
J. Bacteriol.
176,
2543-2550
subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli.
J. Bacteriol.
160,
1055-1060
