Arch Microbiol (1992) 158:335 343

Archives of

Hicroldology

9 Springer-Verlag 1992

Genes for a second terminal oxidase in

Bradyrhizobiumjaponicum

Michael Bott, Oliver Preisig, and Hauke Hennecke Mikrobaologisches Institut, Eidgen6ssische Technische Hochschule, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Ztirich, Switzerland

Recewed March 27, 1992/AcceptedJune 23, 1992

Abstract. Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa3type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicurn DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa 3 but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved. A remarkable feature of the DNA seqence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B.japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid. Key words" Bradyrhizobium japonicum - Branched respiratory chain - Cytochrome oxidase genes - Heme/ copper oxidases

Bradyrhizobiurn japonicum is a gram-negative soil bacterium with the capability to induce the formation of root Correspondence to." H. Hennecke Abbreviations: ORF, open reading frame; TMPD, N,N,N',N'-

tetramethyl-p-phenylenediamine

nodules on soybean in which the bacteria, then called bacteroids, live as true endosymbionts and fix molecular dinitrogen. Preferably, oxygen is used as the terminal electron acceptor in an exclusively respiratory energy metabolism in this bacterium. However, the cells may face extremely different ambient oxygen concentrations in the free-living versus endosymbiotic life styles. Within the infected nodule tissue, the oxygen concentration (3 to 30 nM) is by a factor of 104 to 105 lower as compared with standard aerobic conditions (250 gM) (Layzell et al. 1990; Witty and Minchin 1990). The usual way of bacteria to cope with such extremes is by induction of different respiratory chains terminated by oxidases with different affinities for 02. In a comparative spectroscopic analysis, Appleby (1969a, b) first showed that the hemoprotein pattern of aerobically cultured B. japonicum cells differed from that of root nodule bacteroids. In particular, cytochrome aa 3 and cytochrome o, whose oxidase function in aerobic cells was demonstrated by photochemical action spectra, were absent in bacteroids. Keister and Marsh (1990) showed that several B.japonicum strains retain cytochromes o and aa 3 even during symbiosis but it is unknown whether these oxidases are functional in the root nodule environment. In the past few years, genetic approaches were used to identify individual components of the B.japonicum respiratory chain. The electron-transfer proteins whose genes were cloned, sequenced and mutagenized include the cytochrome bc 1 complex (fbcFH genes; Th6ny-Meyer et al. 1989), subunit I of the cytochrome aa3 complex (coxA gene; Bott et al. 1990; Gabel and Maier 1990), a novel membrane-anchored 20 kDa cytochrome c, herein called cM (cycM gene; Bott et al. 1991), a soluble cytochrome css2(cycB gene; Rossbach et al. 1991), and a soluble cytochrome c555(cycc gene; Tully et al. 1991). Based on mutant phenotypes, the existence of at least three distinct electron transport chains was postulated. The one most firmly established is active in aerobiosis and consists of the cytochrome bc 1 complex, cytochrome cM and the aa3-type cytochrome c oxidase. This electron transport chain differs from the similar mitochondrial respiratory chain by the fact that the membrane-anchored cytochrome cM replaces a soluble cytochrome c. Since

336

cytochrome bc~ mutants were stillcapable of performing aerobic growth, the cytochrome o oxidase identified by Appleby (1969b) was proposed to be a ubiquinol oxidase that terminates a cytochrome c-independent aerobic respiratory chain. However, this conclusion may have to be modified because Frustaci et al. (1991) reported recently that a B.japonicum strain defective in heine biosynthesis (MLG1; Guerinot and Chelm 1986) showed oxygen-dependent growth in rich medium in the apparent absence of cytochromes. Although their results were in some parts controversial, the existence of a third aerobic pathway composed exclusively of nonheme proteins has to be considered. The only known structural genes for cytochromes whose disruption proved to be fatal for symbiosis were the fbcFH genes for the cytochrome bq complex (Th6nyMeyer et al. 1989). Therefore, a microaerobic respiratory chain was predicted in which the electrons are passed from the bc~ complex to a novel terminal oxidase, possibly the enigmatic high-affinity oxidase (Williams et al. 1991). Cytochromes c552 and c55s which once had been suggested as candidates for electron transfer between these components were meanwhile shown to be dispensable for symbiosis (Appleby et al. 1991 ; Rossbach et al. 1991; Tully et al. 1991). Hence, neither the bacteroid oxidase nor its immediate electron donor have been identified thus far.

Table 1. Bacterial strains and plasmids used in this work

Strain/plasmid

In this paper, we report on the cloning and sequencing of a novel gene cluster (coxMNOP) from B.japonicum which encodes a heine/copper type terminal oxidase, and we describe the phenotypic properties of a coxN mutant strain.

Materials and methods

Bacterial strains and plasmids All of the bacterial strains and plasmids used in this work are listed in Table 1. Bradyrhizobiumjaponicum 1lOspc4 is a spectinomycinresistant derivatwe of strain 3 I l b l l 0 (U.S. Department of Agriculture, Beltsville, Md.) and is called the wild-type throughout this paper.

Media and growth of cells B.japonieum strains were grown aerobically at 28 ~ in peptonesalts-yeast extract (PSY) medium (Regensburger and Hennecke 1983). Antibiotics were added at the following concentrations (in ggml-1): chloramphenicol, 10; kanamycin, 100; spectinomycin, 100; streptomycin, 100; and tetracycline, 60. In PSY agar plates, the tetracycline concentration was raised to 120 gg ml- ~. Escherichia coli was routinely grown at 37 ~ in Luria-Bertani medium (Sambrook et al. 1989). E. eoli strain JM101 was grown as described in the Amersham protocol (1984). Antibiotics were added at the following concentrations (in ~g ml ~): ampicillin, 100; streptomycin, 30; and tetracycline, 10.

Relevant characteristics

Source or reference

DH5e

supE44 AlacU169(~bSO lacZAM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1

JM101

supE thi A (lac-proAB) F'[traD36 proAB + laclqZA M 15] Sm~Spr; hsdR (RP4-2 kan :: Tn7 tet :: Mu, integrated in the chromosome)

Bethesda Research Laboratories, Inc., Gaithersburg, Md., USA Messing 1983

E. coil

$17-1 B. japonicum 110spc4 3430

Spr; referred to as "wild-type" SprSm~; coxN:: (2 (0.25 kb deletion and f2 insertion in coxN) Sp~KmrSm~; coxA :: Tn5 Sp~Km~Smr; fbcH:: Tn5

COX132 2800 Plasmids Sequencing vector M13mp18 Sequencing vector M13mpl9 pBluescript KS + Ap r, cloning vector pSUP202X pHP45f~ pRJ3422" pRJ3430 pRJ3431

AprTcrCmr; mob + (oriTofRP4) XhoI linker insertion in EcoRI site AprSmrSp~; source of f~ fragment Ap r (pBluescript KS +): 5.4 kb XhoI insert carrying B japonicum coxMNOP genes Apr (pBluescript KS + ) Sm~Spr; 0.25 kb BstEII fragment from pRJ3422 replaced by 2.0 kb f~ fi-agment from pHP45f~ AprTCCm s (pSUP202X) Smr; 7.25 kb ZhoI insert of pRJ3430 in pSUP202X

a The insert of this plasmid is shown in Fig. 1

Simon et al. 1983

Regensburger and Hennecke 1983 This work Bott et al. 1990 Th6ny-Meyer et al. 1989 Norrander et al. 1983 Norrander et al. 1983 Stratagene, La Jolla, Calif., USA C. Kiindig, ETH Z/irich Prentki and Krisch 1984 This work This work This work

337 exhibited a completely different restriction pattern indicating the presence of another gene homologous to ctaD but different from coxA. Therefore, a 5.4 kb XhoI fragment of this recombinant was subcloned in pBluescript KS + resulting in plasmid pRJ3422 (Fig. 1). After restriction site mapping the sequence analysis was started at the ctaD-homologous region which had been localized via hybridization; finally, the D N A sequence of the complete 5.4 kb XhoI fragment was determined. The nucleotide sequence obtained (Fig. 2) was analyzed with the U W G C G computer program 'CodonPreference' using the codon frequency table obtained for the B.japonicum group III genes which include all genes except the nod genes and the NifA-regulated genes (Ramseier and G6ttfert 1991). Four complete and one incomplete open reading frame (ORF) were detected which showed a strongly biased codon usage and were present on the same D N A strand; the four complete ORFs were named coxM, coxN, coxO, and coxP. In addition, a second incomplete O R F was recognized at the T-end of the opposite D N A strand (Fig. 1). The c o x M gene started with an A T G at position 933 and stopped at position 1764 with a T A G codon. The predicted c o x M gene product (277 amino acids, Mr 31,327) shared homology with subunit II of cytochrome c oxidases. Sixty-seven nucleotides downstream of c o x M the coxN gene started with an A T G (position 1834) and was terminated with a T G A stop codon at position 3607. The predicted coxN gene product (591 amino acids, Mr 65,653) exhibited about 40% identity with subunit I of cytochrome c oxidases. Based on the codon preference plot, the G T G at position 3606 rather than the A T G at position 3735 was proposed to be the start codon of the next gene (coxO) which thus overlapped with the stop codon of coxN. The coxO stop codon T A G was located at position 4311. The predicted coxO gene product (235 amino acids, M r 25,612) shared homology with subunit III of cytochrome oxidases. The coxP gene started 15 nucleotides downstream of coxO with an A T G at position 4329 and stopped at position 5049 with a TGA. Remarkably, the predicted coxP gene product (240 amino acids, Mr 27,364) was also homologous to subunit III of cytochrome oxidases. The start codons of each of these four cox genes were preceded by putative ribosome binding sites at appropriate distances (Fig. 2). Because of the few nucleotides separating coxM, coxN, coxO, and coxP, these genes most probably form an operon. Data bank searches were performed with the incomplete ORF1 and ORF2 gene products, but no proteins with significant homology were found. Both proteins seem to have transmembrane helices which are underscored in Fig. 2.

Recombinant D N A work For routine work with recombinant DNA, established protocols were used (Sambrook et al. 1989). Lambda DNA was isolated as described by Grossberger (1987). Chromosomal DNA from B. japonicum was prepared essentially as reported by Hahn and Hennecke (1984). DNA sequence analysis Appropriate restricUon fragments of plasmid pRJ3422 were cloned into the M13 vectors mpl8 and mpl9 using E. coli JM101 as host strain. The sequence was determined by the dideoxynucleotide chain termination method (Sanger et al. 1977). The protocols and equipment for automated DNA sequencing were applied (Sequencer 370A and M13-specific fluorescent primers from Applied Biosystems, Foster City, Calif., USA). Computer-assisted DNA and protein sequence analyses and alignments were performed by using the Genetics Computer Group of the University of Wisconsin (UWGCG) and the PC GENE (Genofit, Geneva, Switzerland) software packages. Marker exchange mutagenesis A 0.25kb BstEII fragment of pRJ3422 located within the coding region ofcoxNwas replaced by a 2.0 kb Sinai fragment (~qfragment) of pHP45f~ resulting in plasmid pRJ3430. The 7.15 kb XhoI insert of pRJ3430 was cloned into the suicide vector pSUP202X resulting in plasmid pRJ3431 which was transferred via conjugation into the B.japonicum wild-type using E. coil $17-1 as donor. Exconjugants were selected for streptomycin resistance and screened for tetracycline sensitivity to exclude cointegrates. To confirm the genomic structure of the mutants, chromosomal DNA of selected clones was analyzed by appropriate Southern blot hybridizations. Phenotypic analyses N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity, in vivo differencespectra (reduced minus oxidized), and plant infection tests with soybean (Glycine max L. Merr. cv Williams) were performed as described previously (Bott et al. 1990). Results

Cloning and sequencing of genes for a second terminal oxidase h7 B.japonicum In a previous study, a lambda E M B L 4 library of Bradyrhizobiumjaponicum genomic D N A was hybridized with a fragment of the subunit I gene (ctaDI) from Paracoccus denitrificans cytochrome aa 3 in order to identify genes for terminal oxidases. Five positive recombinants were detected, three of which gave strong hybridization signals. Two of these lambda clones showed overlapping restriction patterns and contained the coxA gene which codes for subunit I of the aa3-type cytochrome c oxidase (Bott et al. 1990). The third lambda clone

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339 PhenoO'pic analysis of a coxN mutant strain

In order to possibly characterize the function of the e o x M N O P gene products in the respiratory metabolism of B. japonicum, the coxN mutant strain 3430 was constructed by marker exchange mutagenesis as described in 'Materials and methods'. Several phenotypic tests were carried out with strain )430 and with appropriate control strains: (i) Soybean nodules induced and infected by strain 3430 were unaffected in symbiotic nitrogen fixation as measured by acetylene reduction (Fix + phenotype). Thus, the enzyme encoded by the c o x M N O P genes is not essential for the establishment of a functional symbiosis and apparently is not identical with the proposed high-affinity terminal oxidase active in bacteroids. (ii) Whole cells of B.japonicum 3430 grown aerobically on PSY plates exhibited TMPD oxidase activities like the wild-type. By contrast, a cytochrome aa 3 mutant (B.japonicum COX 132) showed a strongly reduced activity, and a cytochrome bcl mutant (B.japonicurn 2800) was completely negative. Since the TMPD assay identifies cytochrome c-dependent respiratory chains, the positive phenotype of B.japonicum 3430 can be explained in two ways: either, the c o x M N O P genes encode a ubiquinol oxidase rather than a cytochrome c oxidase, or, if the latter is the case, their expression under the conditions employed is negligible as compared with the cytochrome aa 3 genes. (iii) Growth of B.japonicum 3430 under aerobic conditons in liquid PSY medium was compared with that of the wild-type and COX132. The only difference observed for B. japonicum 3430 was a slight delay in the lag phase, whereas the maximal growth rate (g = 0.116 h 1) and the final optical density (ODsso = 1.2) were similar to the wild-type. In the case of B.japonicum COX132, the final OD55o (0.9 to 1.0) was markedly lower. (iv) In vivo difference spectra (dithionite-reduced minus air-oxidized) of aerobically grown cells showed no differences between the wild-type and mutant strain 3430. In conclusion, the phenotypic analyses with B.japonicum strain 3430 did not yet uncover a function for the c o x M N O P gene products. For unknown reasons, our attempts to construct a coxA-coxN double mutant of B. japonicum have so far been unsuccessful.

Discussion We report here the genetic analysis of a DNA fragment from Bradyrhizobium japonicum which was originally identified and cloned via heterologous hybridization using a fragment of the subunit I gene (ctaDI) from Paracoccus denitrificans cytochrome c oxidase. A cluster of at least four genes was identified (coxMNOP) whose

Fig. 2. Nucleotidesequence of the 5.4 kb XhoI fragment.The region covering ORF2 is shown on both strands, whereas for the region covering coxMNOP and ORF1 only the nontranscribed DNA strand asshown.Presumptiveribosomebindingsites are overscored, putative transmembrane helices in the ORFI and ORF2 proteins are underlined

predicted gene products were homologous to subunit I (CoxN), subunit II (CoxM), and subunit III (CoxO, CoxP) of heme/copper terminal oxidases. This superfamily not only includes mitochondrial and bacterial cytochrome c oxidases but also an increasing number of bacterial quinol oxidases (Saraste et al. 1991a; Gennis 1991). It is interesting to note that the operonal structure, in which the subunit II gene is the first gene, followed by the subunit I and III genes, is conserved in many bacteria. In the following sections, the features that are either common or distinct in the cytochrome c and quinol oxidase groups will be discussed in relation to the c o x M N O P gene products. The c o x M gene product

The predicted CoxM polypeptide (277 amino acids, M r 31,327) exhibited about 20% sequence identity to subunit II of heme/copper oxidases. Figure 3 shows an amino acid sequence alignment of CoxM with subunit II from P. denitr~'cans and Bacillus subtilis cytochrome c oxidase (cytochromes aa 3 and caa3, respectively) and from the Escherichia coli ubiquinol oxidase cytochrome o. Unlike most other bacterial subunit II homologs, a third transmembrane spanning segment (overscored in Fig. 3) rather than a signal peptide seems to be responsible for the correct topology ofCoxM (Chepuri and Gennis 1990; Saraste 1990). The carboxy-terminal, periplasmic part of subunit II is diagnostic for the distinction between cytochrome c oxidases and heme/copper type quinol oxidases. In all of the former proteins, subunit II contains a Cu A prosthetic group bound by two cysteine and two histidine residues which are strictly conserved; in the latter proteins, Cu A as well as its ligands are missing (Chepuri et al. 1990; Lauraeus et al. 1991; Puustinen et al. 1991; Ltibben et al. 1992; Santana et al. 1992). The amino acid sequence derived from c o x M contains all of the four Cu A ligands (marked with circles in Fig. 3) as well as three of the four conserved carboxylic acid residues proposed to be involved in cytochrome c binding (marked with filled triangles in Fig. 3). This suggests that the CoxM protein contains a Cu a prosthetic group and is part of a cytochrome c oxidase. The coxN gene product

The predicted CoxN protein (591 amino acids, M~ 65,653) was homologous to subunit I from heme/copper terminal oxidases. The identity to the corresponding proteins from B. japonicum cytochrome aa 3 (CoxA), P. den# trificans aa 3 (CtaD), B. subtilis caa 3 (CtaD) and E. coli cytochrome o (CyoB) was calculated to be 43%, 40%, 40%, and 35%, respectively. Figure 4 shows an amino acid sequence alignment of these proteins which are strongly hydrophobic and contain at least 12 transmembrane helices (overscored in Fig. 4). Subunit I catalyzes the four-electron reduction of oxygen to water and plays the central role in the proton pumping activity of the enzyme. Three prosthetic groups have been assigned to subunit I: a hexacoordinated low-spin heine, a pentacoordinated high-spin heine and a copper ion (CUB)

340 : : : : : : : : : : : : : : = : :

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BJ-COXM PD-CTAC BS-CTAC EC-CYOA

MAVALI LLLIAIGSVLFHLFS PWWWTPIATNWGYI DDTIN .......... ITFWI TGFVFTAVI L MA IATKRRGVAAVMS LGVATMTAVPA~AQDVLG DL PVI GKPVNGGMNFQPAS S PLAHDQQWLDHFVLY I I TAVTI FVC L MVKHWRLI LLLALVPLLLSGCGKPFLSTLKPAGEVADKQYDLTVLSTLI MVVVVAVVSV MRLRKYNKSLGWLSLFAGTVLLSGCNSA~LDPKGQIGLEQRSLI LTAFGLMLIVVI PAI LMA

BJ-COXM PD-CTAC BS-CTAC EC-CYOA

FMAY CVFRFH H K - - - EGRQAAYNPENKKLE - -WWL - SVGTGVGVAAMLA PG LVVWHQFVTV ...... PADATEVEI-MGQ LLLI C IVRFNRR- - -ANPVPARFTHNTPI E V I W T L V P V L I L V A I G A F S - -- L P I L F R S Q E M P N D - - p- - D L V I K A - I G H I FFYVIVRFRRSRVGENTI P K Q V E G N K F L E I T W T V I PI L L L I I L V I P V V L Y T L E L A D T S P M D K K G R K A E D A L V V N V R A N L VGF- -AWKYRASNKDAKYS PNWSH SNKVEAVVW - P ILI I I FLAVLTWKTTHALEPSKPLAHD .... EKPITIEV-VSM

BJ-COXM PD-CTAC BS-CTAC EC-CYOA

QWQWSFRLPGKDGRLGTSDVRNI S PENPMGLNRDDPHGQDDVVI ENGDLHLPIGKPVKVLLRSVDVLHDFYVPEFRAKMD QWYWSYEY PNDGVAFDALMLEKEA ...... LADAGY S EDEY L LATDNPVVVPVGKKVLVQVTATDVI HAWTI PAFAVKQD YW-WEFEYPDYG .............................. I I TSQELIVPTDQRVYFNLKASDVKHSFWI PSVGGKLD DWKWFFIYPEQG .............................. IATVNE IAFPANTPVYFKVTSNSVMNSFFI PRLG SQIY

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MVPGMVTYFWI ...... RPI RTGTFDVLCAELCGAAHYQMRAKVIVEAESDYHAWLEQQKTFAGLSGRNAVVRAKYNS AVPGRIAQLWF ...... SVDQEGVYFGQC SELCG INHAYMPIVVKAVSQEKYEAWLAGAKEEFAADAS DYLPAS PVKLA TNTDNENKFELTFDSKRSKEAGDMFFGKCAELCGPSHALMDFKVKTMSAKEFQGWTKEMKN- YKSTAESHLAKQGEELF AMAGMQTRLHLI ..... ANEPGT- YDG I SASY SGPGFSGMKFKAIATPDRAAFDQWVAKAKQS PNTMgDMAAFEKLAAPS

274 294 266 258

BJ-COXM PD-CTAC BS-CTAC EC-CYOA

GDD W SAE V KEKNCLSCHAVEPNDKRAEAARTAPNLATFGERTKVAGVKEANKENVKAWLKDPDS EYNQVEYFSNVKPDLFADVINKFMAHGKSMDMTQPEGEHSAHEGMEGMDMSHAESAH

277 297 346 315

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356

Fig. 3. Alignment of amino acid sequences from subunit II of heme/copper type terminal oxidases. The sequences are from Bradyrhizobium japonicum CoxM (this work), Paracoccus denitrificans CtaC (Raitio et al. 1987), Bacillus subtilis CtaC (Saraste et al. 199tb), and Escherichia coli CyoA (Chepuri et al. 1990). Asterisks indicate identical amino acids, conservative substitutions are marked by dots. Confirmed (PD-CTAC) and proposed signal peptides are underlined. Potential transmembrane helices identified

with the program SOAP (PC GENE software package by Genofit, Switzerland)for B. japonicum CoxM are overscored. The carboxylic acid residues proposed to be involved in the binding of cytochrome c are marked by filled triangles. The circles indicate the presumed cysteine and histidine ligands for the CuA prosthetic group. In the cytochrome c domain found at the C-terminus of B. subtilis CtaE, the cysteines involved in covalent heme attachment and the berne iron axial ligands are marked by open triangles

that forms a binuclear center together with the high-spin heine iron. Six strictly conserved histidine residues (marked with open triangles in Fig. 4), all of which are present in CoxN, serve as ligands for the heme irons and CuB. A seventh histidine residue previously proposed to be invariant (marked with a filled triangle in Fig. 4) is replaced by glutamine-395 and serine-407 in the B. japonicure CoxA and CoxN proteins, respectively. In sitedirected mutagenesis studies with E. coli cytochrome o, the corresponding histidine-411 was definitively shown not to be a ligand of either the heme irons or CuB (Minagawa et al. 1992; Lemieux et al. 1992). However, Haltia (1992) recently indicated an involvement of this residue in manganese binding. For several reasons the heme groups bound to CoxN may be of the heme O type (Puustinen and Wikstr6m 1991) rather than of the heine A type. (i) Reduced-minusoxidized difference spectra of aerobically grown cells of the e o x N mutant strain 3430 retain the features typical for cytochrome aa3 (peak at 603 nm, shoulder at 445 nm) whereas spectra of the coxA mutant strain COX132 clearly have lost them (Bott et al. 1990). (ii) The B. japonicure wild-type strain used in this study does not express an aa3-type oxidase in symbiosis (Keister and Marsh 1990). (iii) A tyrosine residue (marked with a circle in Fig. 4) which is invariant in aa3-type oxidases and which has been proposed to form a hydrogen bond with the formyl group of the low-spin heme A (Holm et al. 1987) is absent in E. coli cytochrome o as well as in the

B.japonicum CoxN protein. The E. eoli heme O was

recently shown to differ from heine A only by the replacement of the formyl group by a methyl group (Puustinen and Wikstr6m 1991). (iv) Photochemical action spectra revealed the simultaneous presence of an aa3-type and of an o-type oxidase in aerobically grown cells of B. japonicum strain 505 (Appleby 1969b). The coxO and coxP gene products

The CoxO protein (235 amino acis, calculated Mr 25,612) and the CoxP protein (240 amino acids, calculated M r 27,364) were homologous to each other (26% identity) and to subunit III of heme/copper terminal oxidases. Figure 5 shows an alignment of CoxO and CoxP with the corresponding subunits from P. denitr~'cans (CtaE, 23% and 25% identity, respectively), B. subtilis (CtaE, 19% and 30% identity, respectively) and E. coli (CyoC, 23% and 32% identity, respectively). Like subunit I, subunit III is a strongly hydrophic protein with five to seven transmembrane helices. There are two conserved carboxylic acid residues present in subunit III which are located within proposed transmembrane helices (marked with triangles in Fig. 5). In cytochrome c oxidases, the N-terminal one is a glutamic acid residue with a high reactivity towards covalent modification by dicyclohexylcarbodiimide (DCCD; for references see Haltia et al. 1991), and the C-terminal one is an aspartic acid residue. In E. coli CyoC as well as in B.japonicum CoxP, the

341

BJ-COXN BJ-COXA PD-CTADI BS-CTAD EC-CYOB

MVDVPYDRIADIPPAEVPDVELYHPRSWWTRYVFSQDAKVIAIQYSLTASAIGLVALVLSWLMR MATSAAAHGDHAQDHGHDEHAHPTGWRRYVYSTNHKDIGTMYLIFAVIAGVIGAAMSIAIR MSAQISDSIEEKRGFFTRWFMSTNHKDIGVLYLFTAGLAGLISVTLTVYMR MLNALTEKRTRGSMLWDYLTTVDHKKIAILYLVAGGFFFLVGGIEANFIR MFGKLSLDAVPFHEP••M•TIAGIILGGLALVGLITYFGKWTYLWKEWLT•VDHKRLGIMYIIvAIvMLLRGFADAIMMR

64 61 51 50 80

BJ-COXN BJ-COXA PD-CTADI BS-CTAD EC-CYOB

LQLGFPGTFSFI ................ DANQYLQFITMHGMIMVIYLLTALFLGGFGNYLIPLMVGARDMVFPYVNMLS AELMYPGVQIF ................ HETHTYNVFVTSHGLIMIFFMVMPAMIGGFGNWFVPLMIGAPDMAFPRMNNIS MELQHPGvQYMcLEGMRLVADAAAECTPNAHLWNVVvTYHGILM•FFvVIPALFGGFGNYFMPLHIGAPDMAFPRLNNLS IQ . . . . . . . . . . . . . . . . L A K P E N A - F L S A Q A Y N E V M T M H G T T M I F L A A M P L L F A - L M N A V V P L Q I G A R D V S F P F L N A L G SQ . . . . . . . . . . . . . . Q A L A S A G E A G F L P P H H Y D Q I F T A H G V I M I F F V A M P F V I G - L M N L V V P L Q I G A R D V A F P F L N N L S

BJ-COXN BJ-COXA PD-CTADI BS-CTAD EC-CYOB

YWVYLLAVLVLASAFFVPGGP .... TGAGWTLYPPQAILSGTPGQDWGIVLMLSSLILFIIGFTMGGLNYVVTVLQARTR FWLLPASFGLLLMSTFVEGEPGANGvGAGWTMYvP---LSSSGHPGPAVDFAILSLHLAGASSILGAINFITTIFNMRAP YWLYvCGvSLAIASLLSPGGSDQPGAGvGWVLYPP---LSTT-EAGYAMDLAIFAvHvSGATSILGAINIITTFLNMRAP FWLFFFGGIFLNLSWFLGGAPD ..... AGWTSYASLS--LHS--KGHGIDFSILGLQISGLGTLIAGINFLATIINMRAP FWFTVVGVILVNVSLGVGEFAQ ..... TGWLAYPPLSGIEYS--PGVGVDYWIWSLQLSGIGTTLTGINFFVTILKMRAP

BJ-COXN BJ-COXA PD-CTADI BS-CTAD EC-CYOB

GMTLMRLPLTvWGIFTATvMALLAFPALFVGSVMLLLDRLLGTSFFMPTLvEMGQLSKYGGGSPLLFQHLFWFFGHPEvY GMTLHKMPLFVWSILVTVFLLLLSLPVLAGAITMLLTDRNFGTTFFAPD .......... GGGDPVLFQHLFWFFGHPEVY GMTLFKVPLFAWAVFITAWNILLSLPVLAGGITMLLMDRNFGTQFFDPA .......... GGGDPVLYQHILWFFGHPEVY GMTYMRLPLFTWTTFVASALILFAFPPLTVGLALMNLDRLFGTNFFNPEL .......... GGNTVIWEHLFWIFGHPEVY GMTMFKMPVFTWASLCANVLIIASFPILTVTVALLTLDRYLGTHFFTNDM .......... GGNMMMYINLIWAWGHPEVY

v

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IVALPAFGIvSDLISTHARKNIFGYR•MvWAIVAIGALSFv•rWAHH•YvSGMYPYFGFFFATTTLIIAIPTAIKvYNWVL ILILPGFGMISQIvSTFSRKPvFGYLG•AYAMVAIGGIGFvVWAHH•YTVGMSSATQAYFvAATMvIAvPTGvKIFSWIA MLILPGFGIISHvISTFARKPIFGYLPMvLAMAAIAFLGFIvWAHH•YTAG•SLTQQTYFQ•ATMTIAvPTGIKvFSWIA ILILPAFGIFSEvIPvFARKRLFGYSSMVFAIv-LGFLGFMV••VHHMFTTGLGPIANAIFAvAT•AIAIPTGIKIFNWLL ILILPVFGvFSEIAATF•RKRLFGYT•LvWATvCITvLSFIvWLHHFFTMGAGANVNAFFGITTMIIAIPTGvKIFNWLF

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NDvLGKFHFWvTFLGAYLIFFPMHYLGLLGvPRRYFELGDAAFIPPSAHSLNAFITvvALTVGFAQ•VFLFNLvW--SLF NETLAKAHFWVTFIGVNLVFFPQHFLGLSGMPRRYVDY ...... PDAFAGWNLVSSVGSYISGFGVLIFLYCVIDAF--PEWAGQLHFWNMFIGSNLIFFPQHFLGRQGMPRRYIDY ...... PVEFSYWNNISSIGAYIS-EASFLFFIGI-VFYTLF HETMGKISFVLFFIGFHLTFFIQHFVGLMGMPRRVYTF .... LPGQGLETGNLISTIGAFFMAARVILLLVNV---IWTS NETWGKRAFWFWIIGFFVAFMPLYALGFMGMTRRLSQQ .... IDPQ-FHTMLMIAASGAVLIALGILCLVIQMYVSIRDR *

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522 503 508 485 523

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BJ-COXN BJ-COXA PD-CTADI BS-CTAD EC-CYOB

EGEPSGG-NPW--RATTLEWQTPETPPGHGNWGKQLPIVYRWAYDYSVPGAAQDFIPQNQPPPTGAVQGVAP AKKVPAGDNPWGAGATTLEWTLPSPPPFHQFEVLPRVQ AGKPVNVPNYWNEHADTLEWTLPSPPPEHTFETLPKPEDWDRAQAHR VKGEYv@ADPWHD-GRTLEWTVSSPPPEYNFKQLPFvRGLDPLWIEKQAGHKSMTPAEPvDDIHMPNGSILPLIISFGLF DQNRDLTGDPW-G-GRTLEWATSSPPPFYNFAVVPHVHERDAFWEMKEKGEAYKKP-DHYEEIHMPKNSGAGIVIA•FST

591 541 $54 564 600

BS-CTAD EC-CYOB

VAAFGLLYRSDYAWGLPVIFIGLGITFITMLLRSVIDDHGYHIHKEELPNDDKGVKA IFGFAMIWHIWWLAIVGFAGMIITWIVKSFDEDVDYYVPVAEIEKLENQHFDEITKAGLKNGN

621 663

Fig. 4. Alignment of amino acid sequences from subunit I of berne/ copper type terminal oxidases. The sequences are from Brad)'rhizobium japonicurn CoxN (this work), B.japonicum CoxA (Bott et al. 1990), Paracoccus denitr~'cans CtaDI (Raitio et al. 1987), Bacillus sub tilis (Saraste et al. 1991 b), and Eschermhia coli CyoB (Chepuri et al. 1990). Asterisks indicate identical amino acids, conservative substitutions are marked by dots. Potential transmembrane helices identified for B.japonicum CoxN are overscored, whereas those

found additionally in E. coli CyoB and B. subtihs CtaD are shown in boldface. The six invariant histidines involved in metal binding are marked by open triangles. Thefilled triangle indicates a histidine residue conserved in all known subunit I sequences except B. japonicure CoxA and CoxN. The open circle shows the position of the tyrosine residue proposed to form a hydrogen bond with the formyl group of the low-spin heme A prosthetic group

glutamic acid residue is substituted by aspartic acid while in C o x O both carboxylic acid residues are replaced by neutral amino acids. By site-directed mutagenesis of P. denitrificans CtaE, Haltia et al. (1991) could s h o w that the two carboxylic acids are neither involved in the proton-pumping of the enzyme, nor are they essential for

the function of subunit III in the assembly and stabilization of the complex (Haltia et al. 1989). The presence of two subunit III h o m o l o g s w h o s e genes occur in a tandem arrangement within the same operon is unique in bacteria and m a y be related to the proposed dimeric structure of oxidases (Saraste 1990).

342 = = : = = = = = = = = = = = = = =

BJ-COXO BJ-COXP PD-CTAE BS-CTAE EC-CYOC

NSAVILFLAVIAVIVGWWLSQQRLTAKPWLEAGPIDDFPG MAETALRDTGQVPARLEGWQGISADWASDQ MAHVKNHDYQILPPSIWPFFGAIGA~VMLTGAVAWMKGITFFGLPVEGPWMFLIGLVGVLYVMFGWWADVVNEGETGE MQVQEKFTAETFPASPE MATDTLTHATAHAHEH

BJ-COXO BJ-COXP PD-CTAE BS-CTAE EC-CYOC

TDAMTWPAAKVGLGVFLAVAGSLFTLFISAYSMRMNMVDWRTMPVPK ............... VLWFNTGVLVLSSVALQW RAFKNVSWGKAMMWIFLLSDTFIFSCFLLSYMTARMSTTVPW-PNPSEVFALNIGGKHIPLILIAIMTFILISSSGTMAM HTPVVRIGLQYGFILFIMSEVMFFVAWFWAFIKNALYPMGPDSPIKDGVWPPEGIVTFDPWHLPLINTLILLLSGVAVTW KVTLEGKNNFLGFWLFLGGETVLFASLFATFLAASNSNAGD ........... PPTTEMFDVTLVFIATMLLLTSSLTSVY GHHDAGGTKIFGFWIYLMSDCILFSILFATYAVLVNGTAGG ........... PTGKDIFELPFVLVETFLLLFSSITYGM

BJ-COXO BJ-COXP PD-CTAE BS-CTAE EC-CYOC

ALMAA--RRNDIDGVVvGLLAGGASAIAFLAGQLLAWHQLSDAGY--FMASNPAN---AFFYvITAvHGLHLTGGLVALG AvN-FG-YRRDRvRTAILMLATAAFGATFvSMQAFEWTKLIMEGvRPWGNPWGAAQFGSSFFMITGFHGTHVTIGVIFLI AHHAFV-LEGDRKTTINGLIVAVILGVCFTGLQAYEYSHAAFG ........ LADTVYAGAFYMATGFHGAHVIIGTIFLF AMYHMKNFSFGKMQ--LWLGITILLGAGFLGLEIYEFKHYTHE .... FGFTITSSALGSAFYTLVGTHGAHVAFGLMWIS AAIAM--YKNNKSQVISWLALTWLFGAGFIGMEIYEFHHLIVN ..... GMGPDRSGFLSAFFALVGTHGLHVTSGLIWMA

: : : : : : : : : : : : : : : : :

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*

BJ-COXO BJ-COXP PD-CTAE BS-CTAE EC-CYOC

40 30 78 17 16

: : : : : : : : : : : :

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105 109 158 86 85

: : : : : : : : : : : : : : : : :

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RTTAGVWRHDTGTAEMRL ......... SVELCTIYWHFLLLVWLVLLGLLTGWTDDFVDICRQLLS AIARKVWRGDFDVERRGFFTSRKGYYEIVEITGLYWHFVDLVWVFIFAFFYLW VCLIRLLKGQMTQKQHVGF .......... EAAAWYWHFVDVVWLFLFVVIYIWGR TLMIRNAKRGLNLYTAPKFYV .......... ASLYWHFIDVVWVFIFTVVYLMGMVG VLMVQIARRGLTSTNRTRIMC .......... LSLFWHFLDVVWICVFTVVYLMGAM ***

. .

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178 187 229 160 158

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235 240 274 207 204

**

Fig. 5. Alignment of amino acid sequences for subunit III of heine/copper type terminal oxidases. The sequences are from Bradyrhizobiumjaponicum CoxO and CoxP (this work), Paracoccus denitrifieans CtaE (Raitio et al. 1987), Bacillus subtilis CtaE (Saraste et al. 1991b), and Eseherichia coli CyoC (Chepuri et al. 1990). Potential transmembrane helices identified for B. japonicum CoxO

are overscored, whereas those found additionally in P. demtrificans CtaE are shown in boldface. The two conserved carboxylic acid residues located within presumed membrane spanning segments are marked by filled triangles, the N-terminal one of which has a high affinity towards covalent modification by DCCD

Final remarks

References

As pointed out in the introduction, B. japonicum is able to induce at least three distinct terminal oxidases. T a k i n g into consideration the Fix + p h e n o t y p e of the e o x N m u t a n t and the n o t i o n that the e o x M N O P gene p r o d u c t s f o r m a C u a - c o n t a i n i n g c y t o c h r o m e c oxidase with heine g r o u p s different from heme A, the m o s t likely candidate that could be e n c o d e d by these genes is the o-type oxidase present in aerobically cultured cells. To prove or disprove these assumptions, further sophisticated spectroscopic and biochemical analyses of the c o x N mutant, studies on the conditions that lead to expression of the c o x M N O P genes, and finally purification of the C o x M N O P protein complex are n o w required. O u r prediction that the e o x M N O P genes encode a c y t o c h r o m e c oxidase also raises the question as to the nature of the e y t o c h r o m e c needed as the reducing substrate. One, if not all, of the three soluble c y t o c h r o m e s c identified in B.japonicum m i g h t serve this role (Appleby et al. 1991). The inconspicuous phenotypes o f cycB (c55z) and cycC (c555) mutants, like that of the c o x N m u t a n t reported here, are certainly not inconsistent with that idea (Rossbach et al. 1991; Tully et al. 1991).

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Acknowledgements. We thank our colleagues Gonzalo Acufia for providing the lambda library and Markus Bolliger for the identification of the lambda clone carrying the DNA region investigated in this paper. We gratefully acknowledge Matti Saraste for making available the cloned Paracoccus denitrificans cytochrome aa3 genes. M.B. was a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by grants from the Swiss Nataonal Foundation for Scientific Research and the Federal Institute of Technology, Zfirich.

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