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J Biol Chem, Vol. 274, Issue 48, 33999-34004, November 26, 1999

The Na⁺-F₁F₀-ATPase Operon from Acetobacterium woodii
OPERON STRUCTURE AND PRESENCE OF MULTIPLE COPIES OF atpE WHICH ENCODE PROTEOLIPIDS OF 8- AND 18-kDa^*

Stefan Rahlfs, Sascha Aufurth^§, and Volker Müller^§¶

From the  Institut für Mikrobiologie und Genetik der Georg-August-Universität, Grisebachstrasse 8, 37077 Göttingen, Germany and the ^§ Lehrstuhl für Mikrobiologie der Ludwig-Maximilians-Universität, Maria-Ward-Strasse 1a, 80638 München, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Eight genes (atpI, atpB, atpE₁, atpE₂, atpE₃, atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na⁺-F₁F₀-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F₁F₀-encoding genes from Escherichia coli have homologs in the Na⁺-F₁F₀-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na⁺-ATPase from A. woodii is an enzyme of the F₁F₀ class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE₂ and atpE₃) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE₁ gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE₁ encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F₁F₀-ATPase operons. Furthermore, AtpE₁ is the first duplicated proteolipid ever found to be encoded by an F₁F₀-ATPase operon.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acetobacterium woodii is a strictly anaerobic, homoacetogenic bacterium which relies on a sodium ion potential across its membrane for energy-dependent reactions (1). The sodium ion potential is established by a yet not identified primary pump, connected to the acetyl-CoA pathway (2, 3). The Na+1 established is used as driving force for flagellar rotation as well as ATP synthesis (3-6). The enzyme responsible for Na+-driven ATP synthesis was purified and characterized as a Na+-translocating enzyme (7). It was characterized by immunological methods, inhibitor studies, and by the molecular analysis of the genes encoding subunits , , and  as a Na+-F1F0-ATPase (8-10). A gene encoding subunit c was cloned, and the sequence analysis together with biochemical studies revealed residues potentially involved in Na+ liganding in the proteolipid (11). To date, there are only two thoroughly studied examples in this class of Na+-F1F0-ATPases, the enzymes from Propionigenium modestum (12) and A. woodii.

Interestingly, the purified Na⁺-F₁F₀-ATPase of A. woodii contained only six polypeptides (7). Five were identified as subunits , , , , and c, respectively. Because all bacterial ATPases known to date contain eight non-identical subunits, two subunits (including at least one of the membrane-bound subunits a and b) were apparently missing in the purified enzyme. Nevertheless, the purified enzyme was capable of coupling ATP hydrolysis to Na⁺ transport. A simpler architecture was also postulated for some F₁F₀-ATPases from phylogenetically related clostridia (13-15).

To determine whether the ATPase from A. woodii does indeed contain less subunits than the enzyme from Escherichia coli, an alternative way was chosen. To delineate the subunit composition of the enzyme, we chose a molecular approach taking advantage of the fact that (most) bacterial F₁F₀-ATPase encoding genes are organized in operons (16). Here we describe the cloning and characterization of a DNA region containing eight genes (atpI, atpB, atpE₁, atpE₂, atpE₃, atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All chemicals used were reagent grade and purchased from Merck AG, Darmstadt, Germany. [³⁵S]Methionine was from Hartmann Analytik, Braunschweig, Germany. Antibodies were prepared by Bioscience, Göttingen, Germany.

Organisms and Plasmids-- A. woodii (DSMZ 1030) was obtained from the "Deutsche Sammlung für Mikroorganismen und Zellkulturen" (DSMZ), Braunschweig, Germany, and grown under strictly anaerobic conditions on carbonate-buffered medium supplemented with 0.4% glycine (17). E. coli DH5 (supE44 lacU169 (80lacZM15) hsdr17 recA1 endA1 gyrA96 thi1 relA1 (18)) was grown on Luria broth at 37 °C, E. coli DK6 (F, Str^r, hsdR, minA, minB, purE, pdxC, his, ilv, met (uncB-uncC) (19)) at 30 °C on Luria broth plus 1% glucose. Plasmids used were pBluescript SK and KS (Stratagene), pACYC184 (20), pHSG398 and pHSG399 (21), and pMalc2X (New England Biolabs).

Molecular Procedures-- Chromosomal DNA of A. woodii was isolated by a modified Marmur preparation as described before (11, 22), restricted, size-fractionated by gradient centrifugation, and cloned into pBluescript SK or pACYC184. All procedures used were standard techniques (23). Oligonucleotides were from Life Technologies, Inc. GmbH, Eggenstein, Germany. DNA sequence was determined by the chain termination method of Sanger (24) and analyzed on a VAX computer using the Wisconsin Genetics Computer Group sequence analysis software package, Version 8.1 (University of Wisconsin Biotechnology Center, Madison, WI). Cloning of pAF1 and pSR4 was described before (8, 11). A 2.7-kbp BglII/HindIII fragment from pSR4 that hybridized with a 1.9-kbp EcolI/HindIII fragment was cloned into BamHI/HindIII restricted pACYC184 (pSR184BHV). The cloning of the 5'-terminal region of the atp operon was not possible by conventional methods. However, from Southern blots the presence of a SmaI site 1600 bp upstream of atpE₁ was evident. A degenerated primer carrying the SmaI restriction site (OSmaV, 5'-TT(GT)(GT)(GT)(GT)(GT)CCCGGG-3') and a homologous primer (OAwar, 5'-TCCATCGCCCCAGAAATAAA-3') derived from the 3'-terminal end of the atpB gene were used to amplify by PCR the 5'-terminal region of the atp operon. The resulting 1.6-kbp fragment was cloned into EcoRV restricted pACYC184 and designated pSREVB. After DNA sequencing, PCR was used to verify that this fragment is present as such on the chromosome (data not shown).

Northern Blotting-- For RNA isolation, cells were harvested in the logarithmic growth phase and immediately used or frozen at 70 °C. RNA isolation was done with the "RNeasy^TM Total RNA Kit," Qiagen, Hilden, Germany, as described by the manufacturer.

Heterologous Expression-- pSR184BHV was restricted with EcoRV, and atpE₁, atpE₂, and atpE₃ were cloned into EcoRV restricted pHSG399 (pSR7v). atpE₁ derived by PCR was cloned into pHSG398 (pSRc1v). A 10-bp XhoI linker (CCCTCGAGGG) was inserted into the unique Eco47III site within atpE₁ of pSRc1v, resulting in pSRc1vX. Plasmids were transformed into E. coli DK6, and minicells were isolated essentially as described (25). Gene expression was induced by IPTG. Two identical minicell preparations were pooled, pelleted, and resuspended in buffer (pH 8.0) containing 50 mM Tris-HCl, 50 mM EDTA, 15% sucrose, and 0.3 mg of lysozyme/ml. After incubation on ice for 30 min, cells were centrifuged and resuspended in ice-cold H₂O. The membranes were pelleted by centrifugation, the supernatant containing cytoplasm and periplasm was removed, and the membranes were washed twice. SDS-PAGE, fluorography, and autoradiography was as described (26, 27).

Construction of Plasmid pMal-atpE₁* and Overexpression of AtpE₁*-- Base pairs 154 to 212 of atpE₁ (named atpE₁*) were amplified from chromosomal DNA of A. woodii by PCR using two oligonucleotides which introduced EcoRI and PstI sites, respectively (PatpE₁-3, 5'-ATCGGACAGGAATTCGCGGCC-3'; PatpE₁-4, 5'-TGCTCCTAGCTGCAGAATCAT-3'). The PCR fragment was cloned in pMalc2X, and the resulting plasmid was transformed into E. coli DH5. Cultures were grown in Luria broth at 37 °C, and expression was induced at an A₆₀₀ of 0.5 by adding IPTG to a final concentration of 0.3 mM. After 2 h of growth, cells were harvested, washed, and disrupted at high pressure in a French press. Further purification of the fusion protein was performed as recommended by the manufacturer. For immunization of a rabbit, the entire fusion protein was used.

Immunoblotting-- Cytoplasm and washed membranes of A. woodii were prepared as described previously (7), subjected to SDS-PAGE (26), and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The membranes were blocked for 1 h, washed three times with PBST (140 mM NaCl, 10 mM KCl, 6.4 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.05% Tween 20) for 10 min, and incubated with antisera (4 µg of protein/ml of PBST) for 12 h at room temperature. The membranes were washed again three times with PBST (30 min) and then incubated for 1 h with protein A-horseradish peroxidase conjugate. After three further washing steps (10 min), luminescence was detected using the chemiluminescence blotting substrate from Roche Molecular Biochemicals (Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Organization and transcriptional analysis of the genes coding for the Na⁺-F₁F₀-ATPase from A. woodii-- Previously, an EcoRI fragment containing the 3' end of atpA, the complete atpG, atpD, and atpC genes, as well as the downstream intergenic region, and a following partial open reading frame encoding AlgD was cloned and sequenced (8). We have now cloned the DNA region upstream of this EcoRI fragment on a series of overlapping clones (see "Experimental Procedures"). DNA sequence analysis revealed the presence of eight genes which, based on data base searches, primary sequence alignments, secondary structure predictions, and by comparisons with the experimentally derived N termini of the subunits of the purified protein, encode the previously purified and characterized Na⁺-F₁F₀-ATPase (7). These genes are organized in the order atpI, atpB, atpE₁, atpE₂, atpE₃, atpF, atpH, and atpA (Fig. 1). A strong transcriptional terminator downstream of atpC has been described before (8). To determine whether these genes are organized in an operon, RNA was isolated from fructose-grown cells and probed with a 1.9-kb fragment covering atpE₁-atpA. As can be seen in Fig. 2, the probe hybridized to a fragment of 10 kb. Because the deduced size of the atpI-atpC transcript is 8.035 kb, this experiment gives evidence that atpI through atpC are transcribed, together with non-coding regions upstream and downstream, as one message.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Physical maps of the atp operon and plasmids used in this study. B, BglII; Bs, BstXI; C, ClaI; Ea, EagI; Ec, EcoRI; H, HindIII; P, PstI. A transcriptional terminator is indicated.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Northern blot of A. woodii mRNA probed with atpE1-atpA. RNA was isolated as described and separated by agarose gel electrophoresis. The gel was blotted and hybridized with a fragment covering atpE1-atpA (A). RNA was visualized by UV light after ethidium bromide staining (B). Ethidium bromide was added to only one aliquot of the RNA preparation (lane 2) and the RNA standard (lane 3), but not to the sample in lane 1.

The genes atpI, atpB, atpE₁, atpE₂, atpE₃, atpF, and atpH are predicted to start with an ATG codon, but atpA starts with the rare codon TTG. Every start codon is preceded by well conserved Shine-Dalgarno sequences. Characteristic translational features are shown in Table I. The codon usage of the genes is similar to other organisms, and the GC content of the genes correspond to the GC content of chromosomal DNA of A. woodii.

Properties of the Gene Products-- The properties of the gene products are summarized in Table II. Generally, atpI through atpF code for hydrophobic polypeptides, whereas atpH through atpC code for hydrophilic ones.

AtpI-- atpI codes for a polypeptide with a deduced molecular mass of 14.86 kDa. It is very hydrophobic with two predicted transmembrane spans. 26, 36, and 20% of its residues are identical in AtpI from P. modestum (28), E. coli (29), and Moorella thermoacetica (13), respectively. AtpI was not detected in the purified enzyme (7).

AtpB-- The deduced molecular mass of atpB is 24.48 kDa. It is similar to subunit a from other F₁F₀-ATPases; 45, 41, and 37% of its residues are identical in subunit a of P. modestum (30, 31) E. coli (32), and M. thermoacetica (13), respectively. Subunit a is essential for Na⁺ transport across the membrane, and its structure and function in Na⁺ transport is described elsewhere (33). AtpB was not detected in the purified enzyme (7).

AtpE-- Interestingly, we found three genes downstream of atpB, each encoding a proteolipid-like protein. To exclude any cloning artifacts, different primers were derived from the DNA sequence obtained and used to amplify this region from chromosomal DNA. All controls revealed that there are indeed three copies of atpE present on the chromosome (data not shown). Because this finding is very unusual it is analyzed in more detail below.

AtpF-- atpF codes for a polypeptide with a molecular mass of 20.8 kDa. It has a hydrophobic N terminus. The hydrophilic domain is largely  helical, as revealed by secondary structure predictions. 24, 22, and 28% of its residues are identical in subunit b of P. modestum (30, 31), E. coli (29), and M. thermoacetica (13), respectively, indicating that AtpF is the homolog of subunit b. The similarities are 37, 36, and 42%, respectively. AtpF was not detected in the purified enzyme (7).

AtpH-- The deduced N-terminal sequence of AtpH is identical to the biochemically derived N-terminal sequence (XLVASKYA) of the 19-kDa subunit of the purified enzyme (7). The deduced molecular mass of 20.68 kDa fits well to the experimentally derived value. 29, 22, and 23% of its residues are identical in subunit  of P. modestum (28, 34), E. coli (29), and M. thermoacetica (13), respectively, indicating that the 19-kDa subunit is the homolog of subunit .

AtpA-- The deduced N-terminal sequence of AtpA is identical to the biochemically derived N-terminal sequence (XNL-PEEI) of the 57-kDa subunit of the purified enzyme, which was shown by immunological methods to be the homolog of subunit  (7). This is supported by the molecular data: 53, 60, and 61% of its residues are identical in subunit  of E. coli (29), P. modestum (28, 34), and M. thermoacetica (13), respectively. The deduced molecular mass of AtpA (54.82 kDa) corresponds well to the experimentally derived value.

Three Genes Coding for Subunit c-- The most unusual property which is without precedence in any bacterial species was the finding of three proteolipid-encoding genes (atpE₁, atpE₂, atpE₃) in the F₁F₀-ATPase operon. The DNA sequences of atpE₂ and atpE₃ are nearly identical. Both have 246 base pairs, and only 18 substitutions occurred on the DNA level. The amino acid sequence of the deduced polypeptides is 100% identical, the deduced molecular mass is 8.18 kDa. 69, 42, and 40% of the residues are identical in subunit c of P. modestum, E. coli, and M. thermoacetica, respectively (Fig. 3). The deduced N-terminal sequence matches exactly the biochemically derived sequence (XE(I)LDF(I)K) of the proteolipid detected in the purified enzyme (7). The structure and function of AtpE₂/AtpE₃ and the role of Pro-25, Gln-29, Glu-62, and Thr-63 in Na⁺ transport have been discussed elsewhere (11). However, it should be noted that the previously published sequence was erroneous which led to a protein extended C-terminally by seven residues.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Alignment of the deduced amino acid sequence of the proteolipids of A. woodii with proteolipids of other organisms. Aw1a, residues 1-104 of AtpE1 of A. woodii; Aw1b, residues 105-182 of AtpE1 of A. woodii; Aw2, AtpE2 of A. woodii; Aw3, AtpE3 of A. woodii; Pm, P. modestum (30, 47); Eh, residues 69-156 of the duplicated proteolipid from the V1V0-ATPase of Enterococcus hirae (48); Ec, E. coli (29); Va, Vibrio alginolyticus (49); PS3, thermophilic bacterium PS3 (50); Bm, Bacillus megaterium (51); Sc, Synechocystis PCC6803 (52). The putative Na+-binding site is given in bold letters. The ion specificity of the enzymes is indicated on the left.

Most interestingly, atpE₁ has 546 base pairs, and repeated sequencing did not reveal a stop codon within the sequence of atpE₁. It is more than double the size of atpE₂/atpE₃. The first and second halves are 66% identical on the DNA level, indicating a duplication and subsequent fusion of a precursor gene.

The deduced molecular mass of AtpE₁ is 18.37 kDa with four predicted transmembrane helices arranged in two hairpins. Only 60% of the residues of hairpins 1 and 2 are identical. 70 and 72% of the residues of hairpins 1 and 2, respectively, are identical in AtpE₂/AtpE₃. However, the membrane-buried Na⁺-translocating residue (Glu-62 in AtpE₂/AtpE₃), which is also conserved in H⁺-F₁F₀-ATPases (35, 36), is substituted by a glutamine residue in hairpin 2 (Fig. 3). Apart from Glu-62, the residues Pro-25, Gln-29, and Thr-63 have been proposed to be involved in Na⁺ binding in AtpE₂/AtpE₃ (11); these three residues are conserved in both hairpins. Interestingly, from the multiple alignment it is evident that AtpE₁ contains an enlarged N terminus of 17 residues which is not present in AtpE₂/AtpE₃ (Fig. 3).

Heterologous Expression of atpE₁ in E. coli and Targeting of the Protein to the Membranes-- Although the duplicated proteolipid-encoding gene suggested the presence of a duplicated proteolipid, posttranscriptional modifications had to be excluded. Therefore, atpE₁, atpE₂, and atpE₃ were cloned into an appropriate vector (pHSG399) and expressed in minicells of the ATPase-negative mutant E. coli DK6 in the presence of [³⁵S]methionine (see "Experimental Procedures"). Cell-free extract was separated into membranes and cytoplasm and was subjected to SDS-PAGE and autoradiography. To determine whether the separation of cytoplasm and membranes was successful, the cellular localization of the -lactamase, expressed from pBluescript SK, was analyzed. Pre--lactamase and -lactamase were found predominantly in the membrane fraction and the soluble fraction (containing both cytoplasm and periplasm), respectively, as expected (Fig. 4). When atpE_1, atpE₂, and atpE₃ were expressed, two proteins with apparent molecular masses of 7 and 16 kDa were detected. These proteins were not present in the vector control. The experimentally determined molecular mass of 7 kDa corresponds well to the deduced masses of AtpE₂/AtpE₃, and the experimentally determined molecular mass of 16 kDa corresponds well to the deduced molecular mass of the duplicated proteolipid, AtpE₁. The proteolipids AtpE₁ and AtpE₂/AtpE₃ were found exclusively in the membrane fraction.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Heterologous expression of proteolipids from A. woodii in E. coli. Plasmids were transformed in E. coli DK6, proteins were expressed in the presence of [35S]methionine, and membranes and cytoplasm were separated and subjected to SDS-PAGE and autoradiography. Lane 1, pBluescript SK, cell free extract; lane 2, pBluescript SK, membranes; lane 3, pBluescript SK, cytoplasm; lane 4, pHSG399, cytoplasm; lane 5, pHSG399, membranes; lane 6, pHSG399, cell free extract; lane 7, pSR7v (atpE1 + atpE2 + atpE3), cytoplasm; lane 8, pSR7v, membranes; lane 9, pSR7v, cell free extract; lane 10, pSRc1v (atpE1), cell free extract; lane 11, pSRc1v, membranes; lane 12, pSRc1v, cytoplasm; lane 13, pSRc1vX (atpE1 + linker), cell free extract; lane 14, pSRc1vX, membranes; lane 15, pSRc1vX, cytoplasm.

Because it is known that the proteolipid from A. woodii forms very stable oligomers which are even resistant to boiling in SDS-containing buffer, it had to be excluded that the 16-kDa polypeptide is a dimer of AtpE₂/AtpE₃. Therefore, atpE₁ was cloned separately and, in addition, a frameshift giving rise to a truncated AtpE₁ was introduced by cloning a linker into the coding sequence. When atpE₁ was expressed, only the 16-kDa protein was detected, which again was exclusively found in the membrane fraction (Fig. 4). However, in the construct containing the linker, the 16-kDa protein was no longer present but a 9-kDa polypeptide was found instead in the membranes; the molecular mass of this protein corresponds well to the deduced mass of the truncated protein. These experiments clearly demonstrate that atpE₁ codes for a duplicated proteolipid with an apparent molecular mass of 16 kDa, which is, at least in E. coli, not converted into two 8-kDa proteolipids. Furthermore, the proteolipid from the Gram-positive A. woodii is targeted to the membrane of the Gram-negative host.

Immunological Detection of AtpE1 in A. woodii-- To exclude a hypothetical, A. woodii-specific modification system not present in E. coli, the presence in A. woodii of the duplicated proteolipid, AtpE₁, had to be verified. Therefore, an immunological approach was chosen. To generate an antiserum, atpE₁ was cloned into various vectors and expressed in E. coli as such or as fusion protein. However, overexpression could never be detected; instead, upon induction of transcription growth of the host cells stopped immediately, and the cells lysed. To circumvent this problem, only base pairs 154-212 (named atpE₁*) were fused to malE. This construct was overexpressed in E. coli in high yields. Control experiments revealed that there is no MalE homolog in A. woodii (data not shown) and, therefore, the entire fusion protein was purified and used to immunize rabbits. For the immunological detection of AtpE₁, A. woodii was grown on fructose up to logarithmic growth phase, harvested, and separated into cytoplasm and membranes. After SDS-PAGE, the gel was probed with an anti-AtpE₁* antibody. As can be seen in Fig. 5, the duplicated proteolipid (AtpE₁) was unequivocally present in the membrane fraction. The migration behavior of native AtpE₁ was identical to the heterologously expressed protein, and there was no indication for smaller forms of AtpE₁. The heterologous expression experiments together with the Western blots exclude a posttranslational splitting of AtpE₁ into two 8-kDa proteolipids but gave clear evidence for a duplicated proteolipid in A. woodii, the first ever found to be encoded by an F₁F₀-ATPase operon.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Immunological detection of the duplicated proteolipid, AtpE1, in A. woodii. Cells were grown to late exponential growth phase and harvested by centrifugation. Cytoplasm and membranes were separated by centrifugation, and proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the anti-AtpE1* antibody. The silver-stained gel is shown in panel A and the immunoblot in panel B. Lane 1, membranes; lane 2, cytoplasm. Molecular masses are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

From the data presented here and elsewhere (8), it is evident that the Na⁺-F₁F₀-ATPase operon of A. woodii contains eleven genes. All the genes found in other bacterial species (37) have homologs in A. woodii, and the overall genetic organization in the operons is the same. However, only six polypeptides were previously found in the purified enzyme (7). From the biochemically derived N-terminal sequences of the polypeptides and the deduced sequences, it is now possible to clearly establish the gene-polypeptide correspondence of the purified Na⁺-ATPase from A. woodii (Table III). From this comparison, it is obvious that previous enzyme preparations lacked the duplicated proteolipid (AtpE₁) as well as subunit a (AtpB) and subunit b (AtpF). The same was observed in M. thermoacetica and Moorella thermoautotrophica. Although the encoding genes were present in the atp operon from M. thermoacetica, subunit a and subunit b were not found in purified enzymes (13, 14). Furthermore, antisera against synthetic polypeptides derived from the sequences of subunit a and b of M. thermoacetica did not cross-react with cell free extract of the same organism (13). Since atpB and atpF were transcribed, it was concluded that the messages are not translated in M. thermoacetica. Whether this is also true for A. woodii or whether the subunits a and b were simply lost during the purification procedure remains to be determined. The subunit composition of the enzyme from A. woodii is currently under reinvestigation.

One of the most striking and unique features of the atp operon of A. woodii is the presence of multiple copies of proteolipid-encoding genes. Multiplication of proteolipid-encoding genes have been found before only in V₁V₀-ATPases from Eucarya (38-40) and A₁A₀-ATPases from archaea (41). What could be the selective pressure for multiplication of proteolipid-encoding genes? One has to keep in mind that the subunits of the ATPase are present in different amounts (a₁b₂c₁₂₃₃), and the proteolipid (subunit c) has by far the highest copy number in the complex. Most of our knowledge concerning the regulation of the synthesis of the proteolipid is derived from the paradigm E. coli. There, the proteolipid-encoding gene is part of a polycistronic message, and enhanced synthesis of the polypeptide is achieved by enhancement of translation. In addition, but to a lesser extent, regulation of the mRNA stability contributes to differential gene expression (42, 43). However, an enhancer could not be identified in any of the atpE genes of A. woodii. What other mechanisms could lead to the high copy number of the proteolipid? First, the proteolipid-encoding gene could be part of the polycistronic message but is transcribed, in addition, also separately from its own promoter. This is apparently encountered in the archaeon Methanosarcina mazei (44). Such a mechanism appears to be unlikely in A. woodii, judged from the Northern blots presented here. Second, multiplication of the gene and embedding the copies into the operon would be another way to increase the proteolipid message. This strategy is apparently realized by A. woodii.

Another striking and unique feature is the finding in a F₁F₀-ATPase operon of a gene encoding a duplicated proteolipid. This is without precedence in bacteria. Duplicated proteolipids were, for a long time, seen as an exclusive feature of eucaryal V₁V₀-ATPases (45). In archaea, duplication and triplication of proteolipid-encoding genes with subsequent fusion of the genes was described very recently (41, 46). With the experiments described here we add another argument, now derived from a bacterial species, that multiplied and fused proteolipid-encoding genes are not exclusively present in Eucarya, but also in the other domains of life. This does not necessarily argue against the commonly favored view of evolution of ATPases but could result from horizontal gene transfer which is very often underestimated in natural systems.

It remains to be established whether the duplicated proteolipid is assembled into the enzyme. This question is very crucial in view of the substitution of the membrane-buried carboxylate in hairpin 1 by a glutamine residue. However, it should be noted that the glutamine is capable to ligand Na⁺, the physiological coupling ion in A. woodii. The assembly and oligomeric structure of the proteolipid oligomer of A. woodii is currently under investigation.

	ACKNOWLEDGEMENT

We are indebted to Dr. G. Gottschalk, Göttingen, Germany, for generous support.

	FOOTNOTES

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg "Chemische Aktivitäten von Mikroorganismen" and Mu801/7-2/3).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) U10505.

^¶ To whom correspondence should be adressed: Tel.: 49-89-21806126; Fax: 49-89-21806127; E-mail: v.mueller@lrz.uni-muenchen.de.

	ABBREVIATIONS

The abbreviations used are: _Na⁺, electrochemical sodium ion potential; PAGE, polyacrylamide gel electrophoresis; kbp, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; IPTG, isopropyl-1-thio--D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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