Molecular Cloning, Sequencing, and Physiological Characterization of

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Vol. 267, No. 15, Issue of May 25, pp. 10225-10231,1992

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S. A .

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Molecular Cloning, Sequencing, and Physiological Characterization of the qox Operon from Bacillus subtilis Encoding the aas-600Quinol Oxidase* (Received for publication, October 15, 1991)

Margarida SantanaSp, Frank Kunstli, Marie Frangoise Hullon, Georges Rapoportll, Antoine DanchinSII ,and Philippe Glaser$** From the $Unit& de Regulation de 1’ExpressionGenktique and the lUnite de Biochimie Microbienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15 and the IlEcole Pratique des Hautes Etudes, 46 rue St. Jacques 75005 Paris, France

Bacillus subtilis contains two aa3-typeterminal oxidases (caa3-605and aa3-600)catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H 2 0 (Lauraeus, M., Haltia, T., Saraste, M., and Wikstrom, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of &ox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of &ox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. SUbtiliS, &ox oxidase (aa3-600)is predominant during the vegetative growth and its absence leads to important alterations of the phenotype Of B. SUbtiliS.

ular, the electron transport chains of aerobic or facultatively aerobic procaryotes are most often branched to different terminal oxidases which reducethe molecular oxygen to water (1, 2). Spectrophotometric investigations showed that terminal oxidases are complexed with one of four different hemes. This finding led to a classification of bacterial oxidases into four groups (0,d, al, and aa3) depending upon the heme component they bind (3). Recent work identified two different terminal oxidases in Bacillus subtilis, an aerobic Gram-positive bacterium (4). Like mitochondrial cytochrome oxidase, the analysis of these proteins shows that they both have cytochromes a and a3. However, these oxidases, named caa3-605 and aa3-600 differ in spectral characteristics; the a bands are located at 605 and 551 nm for the first one (the latter band arising from the cytochrome c, which is covalently attached to the enzyme) and at 600 nm for the other one. They also differ in their substrate specificity. The caa3-605 oxidase is a cytochrome c oxidase whereas the aa3-600 oxidase has little or no activity with various cytochromes c as electron donors, but catalyzes the oxidation of quinol (4). The spectral analysis of B. subtilis membranes revealed the existence of a third terminaloxidase containing two cytochrome b components and which is characterized by a CO difference spectrum typical for cytochrome 0 (5). The caa3-605oxidase genes were cloned and sequenced (6). Five genes have been identified named ctaB to ctaF. Genes ctaC to ctaF probably constitute an operon and code for the four subunits of the oxidase, whereas ctaB codes for an assemIn bacteria, respiration is considerably more complex than bly protein. in mitochondria. This complexity could be related to the Analysis of protein sequences of the four caa3-605oxidase ability of bacteria to respond to the availability of energy subunits (6), together with the study of the purified enzyme sources and electron acceptors (most often oxygen). In partic- (4),shows that this enzyme is a member of a large oxidase family. This family includes the mitochondrial a a 3 cytochrome * This work was supported by Unit6 de Recherche Associee (URA) c oxidase, the a a 3 bacterial cytochrome c oxidases like Para1129 and 1300 of the Centre National de la Recherche Scientifique, coccus denitrificans aa3 oxidase (7) and theEscherichia coli bo Grant 91797 from the Direction de la Recherche et1’Enseignement de Supirieur, the Institut Pasteur, and Science Contract SCI* 0211-C quinol oxidase (8). These bacterial oxidases are similar in pumps (Refs. 9 and from the Commission of the European Community. The costs of function (in particular, they act as proton publication of this article were defrayed in part by the payment of 10)) andin protein structure but do not necessarily have the page charges. This article must therefore be hereby marked “aduer- same substrate specificity. Quinol is the substrate for E. coli tisement” in accordance with18 U.S.C. Section 1734 solely to indicate cytochrome bo. Although these bacterial oxidases are structhis fact. turally similar to the mitochondrial enzyme, theycontain The nucleotide sequence(s) reported in thispaperhas been submitted to the GenBankTM/EMBLDataBankwith accession number(s) fewer subunits, which are homologous to the major subunits M86548. of the mitochondrial enzyme (1). Protein structuresimilarities Supported bya JuntaNacionalde InvestigaGGo Cientificae are highly significant for the largest subunit, I, which binds Tecnol6gica fellowship. the two a and a3 hemes (two b hemes in the case of E. coli ** TOwhom correspondence should be addressed Unit6 de Reguquinol oxidase) and the redox-active copper CUB,as well as lation de I’Expression GenGtique, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-45-68-84-41;Fax: 33- for subunit 111, whose function in the oxidase is still controversial (11).Subunit 11, which is thought to bind the redox1-43-06-98-35.

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active copper CuA and the oxidized substrate (reviewed in Refs. 9 and lo), is the least conserved subunit. In the frame of the European project to determine the entire DNA sequence of the B. subtilis genome, the gerB-sacs DNAregionwas assigned to the group from the Pasteur Institute. We identified four genes in this region which probably constitute an operon. A comparison of the translated sequences to known protein sequences revealed significant amino acid similarities to the mitochondrial-type oxidases (e.g. P. denitrificans cytochrome c oxidase, B. subtilis caa3-605 oxidase, and E. coli quinol oxidase). Using reverse genetics, we produced a mutationin the discovered oxidase operon. We used this mutant strainto show that thisoperon codes for the aa3-600 quinol oxidase. Therefore, we propose that aa3-600 enzyme, like caa3-605 enzyme, belongs to the oxidase family exemplified by mitochondrial cytochrome aa3 oxidase and bo quinol oxidase of E. coli.

encoding an a a 3 cytochrome c oxidase, of the genes coding for cytochrome c oxidase of Bacillus PS3, and of the E. coli cy0 operon encoding a cytochrome o quinol oxidase. However, in the lattercases, a fifth gene is foundctaB, preceding ctaC for B. subtilis (6) and cyoE, the last gene of the cy0 operon (8). The product of this fifth gene is presumably involved in the assembly of the complex (6). We found no equivalent of these genes in the vicinity of the newly identified operon. In B. subtilis the ctaB gene product may be necessary to theassembly of both a a 3 oxidases. Indeed, the ctaB gene is separated from the downstream genes by 240 bp andcould beexpressed independently, as pointed out by Saraste andco-workers (6). The nucleotide sequence of the qox operon is presented in Fig. 2. The predicted sequences of the four proteins, QoxA (suII), QoxB (SUI),QoxC (suIII), and QoxD (suIV) are also shown. Physical mapping of the chromosomal region between s a c s and s a c T and the knowledge of X sacPT nucleotide sequence allow us to precisely localize this operon on the MATERIALS AND METHOD$ chromosome at a distance of 30 kb from the s a c s locus and Spectral Analysis-Spectrophotometric studies were carried out 7.5 kb from the sacT gene. The qox operon is transcribed in with mutant and wild-type strains grown in LB medium. To deter- the direction of the replication fork. We identified two potenmine the cytochrome content in the two strains, sodium hydrosulfite tial transcription termination signals, one 782 bp upstream reduced-minus-oxidized difference spectra were performed. These from the qoxA initiation codon ending another putative opeexperiments were carried out at liquid nitrogen temperature (77 K) on the bacterial pellet, using a dual-wavelength scanning spectropho- ron, which has the same orientation as the qox operon, and tometer (LERES) (33). To obtain the CO difference spectra, centri- the second one downstream from qoxD gene. These structures fuged cells were resuspended in MM2 medium containing 0.5% glu- consist of inverted repeats followed by a stretch of 5 thymine cose; spectra were recorded with a Kontron Uvikon 860 spectropho- residues (indicated by arrows in Fig. 2). The 782-bpDNA tometer at room temperature. The scanning speed was 2 nm/s, and region upstream from the qoxA gene may be involved in the 1-cm light path cuvettes were utilized. A stream of CO was bubbled control of the expression of the qox operon, since no other for 1 min through the sample cuvette just before scanning. potential coding sequence is apparent. No sequence with a highdegree of conservation with the different consensus RESULTSANDDISCUSSION sequences of B. subtilis promoters was found in this region. Cloning and Sequence Determinution-We constructed a We have compared this sequence with the upstream sequences chromosomal DNA library in X phage Fix 11, using a partial of the ctaB gene and the men operon (36), respectively, this fill-in strategy (15) in order to sequence the chromosomal last operon being involved in the biosynthesis of menaquiDNA spanning from gerB (314")to s a c s (333") (34).Recom- none, the likely substrate of aa3-600 oxidase. We have found binant X containing DNA fragments of this region were iden- a conserved sequence (Fig. 3 and Fig. 2, underlined). Identitified by hybridization with cloned genes of the gerB-sacs fication of the mRNA end of the men operon shows that this DNA region. X s a c P T was isolated by screening the library sequence corresponds to the men promoter (36). This conwith a probe including the s a c P gene (35). served sequence could bethe binding site of a common tranThe absence of rearrangements between the cloned frag- scriptional regulator for these three loci. ment in X s a c P T and B. subtilis chromosomal DNAwas A putative ribosome binding site for each gene of the qox checked by Southern blot analysis (data not shown). The 18- operon is located four to eight bases upstream from each start kb chromosomal insert from X s a c P T was subsequently se- codon (Fig. 2, double-underline). In the case of qoxA, the quenced on both strands. The complete DNA sequence and initiation codon could not be predicted unambiguously from the entire sequencing strategy will be published elsewhere. the DNA sequence. Fig. 1presents a restriction map of the insert. The s a c T gene Analysis of the Subunit Protein Sequences-In terms of is located at theright end. protein structural similarities, two subfamilies of mitochonPutative coding sequences were identified on both DNA drial type a a 3 oxidases can be distinguished (6). One branch strands and the deduced protein sequences were compared includes proteins like the P. denitrificans oxidase and mitowith known sequences from protein sequence data libraries. chondrial oxidases, while the second branch includes B. subWe found four sequences presenting similarities with the tilis and Bacillus PS3 caa3 oxidases and the E. coli bo quinol subunits of several bacterial oxidases and mitochondrial cy- oxidase (6). In Fig. 4 we aligned protein sequences from the tochrome c oxidases, which seemto be organized in anoperon. four &ox subunits with sequences from subunits of P. deniOur results show that this operon encodes the four subunits trificans oxidase (first subfamily) and with sequences of E . of the aa3-600 quinol oxidase isolated by Lauraeus and coworkers (4). We named these genes qoxA to qoxD, which qox TTTTCATCTTaagcGAtAgATGTgaATAAAGTTggAG specify the subunits 11, I, 111, and IV, respectively (Fig. 1). cta ccTTCATCTTT--GGtAAaATtcaTAaAAAGTTcAca m e n TTTTCpTtTcT--GaAAttAgGTtTATAAtaggtAAG This operon is reminiscent of the B. subtilis cta operon ***** * * ** ~~~

Portions of this paper (including part of "Materials and Methods" and Figs. 1and 2) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. The abbreviations used are: MM, minimal salts medium; LB, Luria-Bertani medium; bp, base pair(s); kb, kilobase(s); su, subunit; ["'S]dATPaS, deoxyadenosine 5'-(~~-thio)triphosphate.

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FIG. 3. Upstream conserved DNA sequences in B. subtilis qox operon, ctaB gene, and men operon. The conserved se-

quences are located 125 bp upstream from the first putative translation initiation codon of qoxA and 122 bp upstream from the translation initiation codon of ctaB. Proposed -35 and -10 regions from the men promoter are underlined twice. Conserved bases in two sequences are in uppercase letters; conserved bases in the three sequences are marked with an asterisk.

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coli quinol oxidase and B. subtilis caa3-605 oxidases (second subfamily). These alignments showed that &ox oxidase (uu3600) belongs to thesecond subfamily. Indeed, compared with the P. denitrificans enzyme, &ox subunit I has two additional C-terminal transmembrane domains. In addition, &ox subunit I11 lacks two other transmembrane domains, as is the case in other oxidases of the second subfamily. Furthermore, the&ox protein may have a fourth subunit equivalent to that found in the oxidases of this group. We emphasize that this group is particularly homogeneous, both at the level of amino acid sequence of the subunits andat the level of genetic organization. In this group, aa3-600, which is an uu3 quinol oxidase, could be considered as an intermediatebetween cau3-605 and E. coli bo quinol oxidase. Indeed, it presentsa higher level of similarity with both other oxidases than these oxidases do with each other. It has been shown, after purification, that the subunitI of the aa3-600 oxidase is slightly larger than subunit I of caa3605 oxidaseencodedby the B. subtilisctu operon (4).In contrast, subunit I1 is slightly smaller than cua3-605 subunit I1 (4).These data are consistent with our results, since it is predicted from the nucleotide sequence that &ox subunit I (QoxB) is 28 amino acids larger, and subunit I1 (QoxA) is 38 amino acids smaller than corresponding the subunits encoded by the cta operon. Subunit I-Among the subunits of &ox oxidase, subunit I (649 residues), encoded by the qoxB gene, presents thehighest degree of similarity. Indeed, thisproteinpresents 51% of amino acid identity with the major subunit of the E. coli cytochrome bo (CyoB), 48% with that of B. subtilis cytochrome c oxidase (CtaD), and40% with thatof P. denitrificuns cytochrome c oxidase (Fig. 4A).The hydropathyprofile of the qoxB gene product shows that it has up to 15 transmembrane spans (Fig. 5 A ) as shown for the cytochrome o complex of E. coli (37). The C terminus of the three proteins QoxB, CyoB, and CtaD contains two more hydrophobic segments as compared with the mitochondrial subunit I. Also, QoxB and the E. coli subunit I have an additional N-terminal hydrophobic segment (see Fig. 5 A ) , lacking in mitochondrial subunit I. Cytochrome c oxidase subunit I containsthe 7 histidine bis-imidazole ligands of the cytochrome a3/CuB center and the A

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... FIG. 4. Alignment of deduced amino acid sequences of subunits from B. subtilis Qox oxidase (Qox), E. coli bo oxidase ( C y o ) ,B. subtilis aa3-605 oxidase ( C t a ) ,and P. denitrificans aa3oxidase ( C O ) . Residues conserved in two sequences are in black bones. Residues conserved in the four proteins are marked by an asterisk and in the three first sequences by a plus sign. The proposed transmembrane domains are indicated by solid lines. A , subunit I. The 7 conserved histidine residues are indicated by arrows. B, subunit 11. The proposed histidine and cysteine ligands for CuA, the carboxylic residues proposed to bind cytochrome c, and the proposed signal peptide cleavage site are indicated by arrows. The aromatic amino acid-rich region is indicated by asterisks. The putative signal peptide cleavage site is indicated by an opentriangle. C, subunit 111. The glutamate residue which binds dicyclohexylcarbodiimide is marked by an arrow. D,subunit IV. Amino acid sequences were deduced from nucleotide sequences (6-8).

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