Sequence analysis, characterization and CO-specific transcription of the cox gene cluster on the megaplasmid pHCG3 of Oligotropha carboxidovorans

Gene 236 (1999) 115–124 www.elsevier.com/locate/gene

Sequence analysis, characterization and CO-specific transcription of the cox gene cluster on the megaplasmid pHCG3 of Oligotropha carboxidovorans Beatrix Santiago, Ulrich Schu¨bel, Christine Egelseer, Ortwin Meyer * Lehrstuhl fu¨r Mikrobiologie, Universita¨t Bayreuth, Universita¨tsstraße 30, 95440 Bayreuth, Germany Received 20 April 1999; accepted 11 June 1999; Received by W. Martin

Abstract Sequence, transcriptional, mutational and physiological analyses indicate that the carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is an integral and unique part of an elaborate CO oxidizing system. It is encoded by the 14.5 kb gene cluster coxBCMSLDEFGHIK residing on the 128 kb megaplasmid pHCG3. The CO dehydrogenase structural genes coxMSL are flanked by nine accessory genes arranged as the cox gene cluster. The cox genes are specifically and coordinately transcribed under chemolithoautotrophic conditions in the presence of CO as carbon and energy source. With the exception of CoxB and CoxK, all deduced products of the cox genes of O. carboxidovorans have counterparts in so far uncharacterized gene clusters of Pseudomonas thermocarboxydovorans, Hydrogenophaga pseudoflava, Bradyrhizobium japonicum, and Mycobacterium tuberculosis. Transposon mutagenesis suggests a function of CoxH and CoxI in the interaction of CO dehydrogenase with the cytoplasmic membrane. The specific functions of the other accessory Cox proteins are difficult to envisage right now, as the polypeptides do not show significant homologies with functionally characterized proteins in the databases. In addition to the clustered cox genes, mutational analyses have identified the genes lon, cycH and orfX which reside on the plasmid pHCG3. The Lon protease, the CycH protein and the unknown orfX gene product have essential functions in the utilization of CO. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Carbon monoxide dehydrogenase; cbb; Chemolithoautotrophy; cyc; hox; lon; Molybdenum hydroxylase

1. Introduction Oligotropha carboxidovorans [ formerly Pseudomonas carboxydovorans (Meyer et al., 1993c)] is a member of the alpha subclass of the Proteobacteria (Auling et al., 1988) and the most intensively studied representative of the carboxidotrophic bacteria. The carboxidotrophic bacteria are characterized by the utilization of carbon monoxide (CO) as a sole source of carbon and energy under aerobic (or denitrifying) chemolithoautotrophic conditions (for a review refer to Meyer et al., 1993a). CO dehydrogenase ( EC 1.2.99.2) from O. carboxidovorAbbreviations: aa, amino acids; bp, base pair(s); Cm, chloramphenicol; kb, kilobase(s); Km, kanamycin; nt, nucleotide(s); PAGE, polyacrylamide-gel electrophoresis; r, resistant; s, sensitive; SD, Shine– Dalgarno sequence; SSC, 0.15 M NaCl/0.015 M Na citrate pH 7.6; 3 Tc, tetracycline; Tn, transposon. * Corresponding author. Tel.: +49-921-552728; fax: +49-921-552727. E-mail address: [email protected] (O. Meyer)

ans is a Se-containing molybdo-iron–sulfur flavoprotein which catalyzes the oxidation of CO with H O, yielding 2 CO , two electrons and two H+ (Meyer et al., 1993a). 2 CO dehydrogenase generates in its membrane-associated state a proton gradient across the cytoplasmic membrane by channelling the electrons formed via cytochrome b into a CO-insensitive respiratory chain (Meyer 561 et al., 1990). The presence of a membrane-bound class I NiFe hydrogenase enables the bacterium to grow with H plus CO under chemolithoautotrophic conditions 2 2 as well (Santiago and Meyer, 1997). The crystal structure of CO dehydrogenase from O. ˚ resocarboxidovorans, which has been solved at 2.2 A lution, shows a dimer of heterotrimers (Dobbek et al., 1999). Each heterotrimer is composed of a 88.7 kDa molybdoprotein (L), a 30.2 kDa flavoprotein (M ), and a 17.8 kDa iron–sulfur protein (S). The molybdoprotein contains the Mo ion and molybdopterin cytosine dinucleotide (MCD) in a 1:1 molar mononuclear complex (Meyer et al., 1993b; Dobbek et al., 1999).

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 24 5 - 0

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B. Santiago et al. / Gene 236 (1999) 115–124

CO dehydrogenase is a prototype of the molybdenum hydroxylase sequence family, which also includes xanthine dehydrogenase/oxidase (Schu¨bel et al., 1995). The CO dehydrogenase structural genes (cox) are clustered in the transcriptional order 5∞ coxMcoxScoxL 3∞ (Schu¨bel et al., 1995). The hydrogenase structural genes (hox) are clustered in the transcriptional order 5∞ hoxShoxL 3∞ (Santiago and Meyer, 1997). The cox and hox clusters flank the cbb gene cluster, which codes for the enzymes of autotrophic CO fixation. The three 2 gene clusters are assembled on a 30 kb DNA segment of the 128 kb megaplasmid pHCG3 of O. carboxidovorans (Santiago and Meyer, 1997). As the CO dehydrogenase structural genes of O. carboxidovorans [coxMSL (Schu¨bel et al., 1995)], Pseudomonas thermocarboxydovorans [cutBCA (Pearson et al., 1994)] and Hydrogenophaga pseudoflava [cutMSL ( Kang and Kim, 1999)] show the same physical and transcriptional arrangement, this seems to be an invariant property of the carboxidotrophic bacteria. CO dehydrogenase is a complex metalloprotein, and the utilization of CO for growth requires several components in addition to CO dehydrogenase, e.g. proteins

involved in the association of the enzyme with the cytoplasmic membrane and the transfer of electrons to the respiratory chain (Meyer et al., 1990). To identify potential accessory genes with essential functions in the metabolism of CO, the 7.8 kb intergenic region between coxL and cbbR as well as the region upstream of coxM were sequenced and characterized by mutational and transcriptional analysis.

2. Materials and methods 2.1. Bacterial strains, plasmids, and growth conditions The strains and plasmids employed are listed in Table 1. Chemolithoautotrophic growth was in the mineral salts medium described (Meyer and Schlegel, 1983) under a gas atmosphere composed of (% vol/vol ) 45 CO, 5 CO and 50 air, or 40 H , 10 CO and 50 air. 2 2 2 For heterotrophic growth the mineral medium was supplemented with 0.2% pyruvate and 0.3% nutrient broth. Escherichia coli was grown in Luria–Bertani medium.

Table 1 Bacterial strains and plasmids used Strain or plasmid Bacteria O. carboxidovorans OM5 TN-81 TN-106 TN-398 TN-424 TN-470 E. coli DH5a S17-1 Plasmids pHCG3 pBluescript I KS+/SK+ pSUP1011 pCDH1 pBS7.6 pBS5.7 pBS2.2 pCAC1 pCAC7 pCAC8 pCAC9 pKT7-9 pTn81 pTn106 pTn398 pTn424 pTn470

Genotype or descriptiona

Reference or source

Wild type, Kans, Tetr Cox−, Cbb+, Hox+, Kr m Cox−, Cbb+, Hox+, Kr m Cox−, Cbb+, Hox+, Kr m Cox−, Cbb+, Hox+, Kr m Cox−, Cbb+, Hox+, Kr m

Meyer and Schlegel, 1978 This study This study This study This study This study

w 80d lacZDM15 endA1 recA1 hsdR17 (r− m+) supE44 rpsL20 thi-1 l− K K gyrA96 relA1 F−1 D(lacZYA-argF ) U169 pro thi hsdR17 (r− m+) recA RP4-2( Tcr::Mu-Kmr::Tn7) K K

Jessee, 1988

128 kb plasmid of O. carboxidovorans OM5 Apr Cmr, Kmr, Tn5 containing mobilization vector a 4.86 kb BamHI–HindIII fragment of pHCG3 in pBluescript I KS+ a 7.6 kb SalI fragment of pHCG3 in pBluescript I KS+ a 5.7 kb SalI fragment of pHCG3 in pBluescript I KS+ a 2.2 kb EcoRV fragment of pHCG3 in pBluescript I SK+ a 5.7 kb EcoRV fragment of pHCG3 in pBluescript I SK+ a 1.7 kb HindIII–BamHI fragment of pHCG3 in pBluescript I SK+ a 2.3 kb SalI–HindIII fragment of pHCG3 in pBluescript I SK+ a 2.9 kb EcoRV fragment of pBS7.6 in pBluescript I SK+ a 5.9 kb SalI fragment of pHCG3 in pT7-5 a 11.4 kb EcoRV fragment of pHCG3 with Tn5 in pBluescript I KS+ a 8.6 kb EcoRV fragment of pHCG3 with Tn5 in pBluescript I KS+ a 9.5 kb ClaI fragment of pHCG3 with Tn5 in pBluescript I KS+ a 8.7 kb ClaI fragment of pHCG3 with Tn5 in pBluescript I KS+ a 8.6 kb EcoRI fragment of pHCG3 with Tn5 in pBluescript I KS+

Kraut and Meyer, 1988 Stratagene Simon et al., 1983 Schu¨bel et al., 1995 Santiago and Meyer, 1997 Santiago and Meyer, 1997 Santiago and Meyer, 1997 This study This study This study This study Kraut and Meyer, unpublished This study This study This study This study This study

Simon et al., 1983

a Apr, ampicillin resistant; Kr , kanamycin resistant; Ks , kanamycin sensitive; Tetr, tetracycline resistant; Cmr, chloramphenicol resistant. m m

B. Santiago et al. / Gene 236 (1999) 115–124

2.2. Recombinant DNA techniques Total bacterial DNA was isolated with Nucleobond AXG20 (Macherey & Nagel, Du¨ren, Germany). Plasmid DNA from O. carboxidovorans or from E. coli was isolated with Nucleobond PC100 or PC500 (Macherey & Nagel, Du¨ren, Germany). E. coli was transformed using a Gene Pulser (Bio-Rad, Mu¨nchen, Germany). Restriction enzymes and modifying enzymes were purchased from Gibco BRL (Eggenstein, Germany). 2.3. DNA sequence analysis Sets of nested deletions were introduced bidirectionally into the cloned region of pCAC1, pCAC7, pCAC8 and pCAC9 by treatment with exonuclease III-S1 nuclease of the Erase-a-Base kit (Promega, Madison, WI ). Sequencing of both strands of cloned DNA with the reverse and the universal primers was performed with the Licor sequencing system (MWG, Ebersberg, Germany). The HUSAR progam package (Deutsches Krebsforschungszentrum, Heidelberg, Germany) was employed for sequence analysis.

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were at 68°C and washes with 0.2×SSC/0.1% SDS at 58°C. 2.5. Analysis of transcription by slot–blot hybridization Total RNA was isolated from mid-log phase cultures of O. carboxidovorans by the method of Arps and Winkler (1986). Total RNA (1 mg) was transferred onto a positively charged nylon membrane (Amersham, Braunschweig, Germany) by slot blotting. After being baked at 120°C for 1 h, the filters were prehybridized, hybridized at 50°C in 50% formamide and 5×SSC with digoxigenin labeled DNA probes, and washed (1×SSC/0.1% SDS at 68°C ). Hybridization signals were detected as luminographs according to the manufacturer’s instructions (Boehringer, Mannheim, Germany) employing Kodak BioMax MS films. The hybridization signals were quantified with the ZeroDScan software (Scanalytics, Billerica, MA). Gene probes used for slot– blot analyses but not documented: coxM, 0.63 kb BamHI–SacII fragment of pCDH1; coxS, 0.56 kb HincII–EcoRV fragment of pCDH1; coxE, 1.1 kb HindIII–SalI fragment of pCAC1; coxF, 1.6 kb SalI– HindIII fragment of pCAC1; coxH, 1.6 kb HindIII– EcoRV of pCAC1.

2.4. Labeling and hybridization of gene probes 2.6. Transpositional mutagenesis Restriction fragment gene probes were labeled with digoxigenin-11-dUTP by use of the DIG DNA Labeling Kit (Boehringer, Mannheim, Germany). Hybridizations

The transposon Tn5 was introduced into O. carboxidovorans on pSUP1011 (Simon et al., 1983) by conjuga-

Fig. 1. Physical and genetic map of the cox region on the plasmid pHCG3 of O. carboxidovorans showing the 30 kb DNA region of pHCG3 which carries the cox, cbb, and hox structural genes (Santiago and Meyer, 1997). Depicted are the subcloned and sequenced fragments, the lengths and the transcriptional order of the identified genes. The closed arrow heads refer to the points of transposon insertion: B, BamHI; V, EcoRV; H, HindIII; S, SalI. The sequences of the cox genes are available from the GenBank nucleotide sequence database under the accession number X82447.

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B. Santiago et al. / Gene 236 (1999) 115–124

tion with E. coli S17-1. Donor cells or recipient cells were harvested by centrifugation at the exponential growth phase, washed in 0.9% NaCl and then mixed in a 1:100 ratio (donor:recipient). Cell suspensions were transferred to membrane filters (0.2 mm pore size) placed on mating medium (heterotrophic mineral medium without antibiotics). The filters were incubated for 36 h at 30°C, washed, and cell suspensions, concentrated in 0.9% NaCl, plated on selective plates, containing Km (500 mg/ml ) and Tet (100 mg/ml ). As a control for spontaneous mutations, the recipient strain was also selectively plated.

3. Results

2.7. Enzyme assay and protein determination

3.1. Sequence and molecular organization of the regions flanking coxMSL

CO dehydrogenase was assayed spectrophotometrically by following the reduction of INT [2-(4-iodophenyl )-3-(4-nitrophenyl )-2H-tetrazolium chloride] with CO employing MPMS (1-methoxyphenazine methosulfate) as a mediator ( Kraut et al., 1989). Protein estimations were done as described before (Santiago and Meyer, 1997).

For sequencing the region upstream of coxM, a 1.7 kb HindIII–BamHI fragment or a 2.3 kb SalI–HindIII fragment of pHCG3 from O. carboxidovorans were inserted into the vector pBluescript yielding the plasmids pCAC7 and pCAC8 ( Fig. 1). For sequencing the region downstream of coxL, a 5.7 kb EcoRV fragment or a 2.9 kb EcoRV fragment of pHCG3 were inserted into the vector

2.8. Western immunoblot analysis Proteins were separated by non-denaturing PAGE (5% stacking gel, 7.5% running gel ) as described before ( Kraut et al., 1989), and then electroblotted onto poly(vinylidene difluoride) membranes. Blots were developed with polyclonal IgG antibodies (0.2 mg/ml ) directed against CoxL and a goat anti-rabbit alkaline phosphate conjugate (Sigma, Deisenhofen, Germany).

Fig. 2. Slot–blot analysis of total RNA isolated from O. carboxidovorans grown under the indicated conditions. 1 mg of purified total RNA was spotted onto nylon membranes for slot–blot analysis employing the indicated specific digoxigenin labeled DNA probes. Results with the following selections of gene probes used for slot–blot analysis are shown: orf7, 0.65 kb AvaI fragment of pCAC8; coxB, 0.95 kb PvuI–HindIII fragment of pCAC8; coxC, 0.86 kb HindIII–PvuI fragment of pCAC7; coxL, 0.69 kb AvaI fragment of pCDH1; coxD, 1.0 kb BsiWI–HindIII fragment of pCDH1; coxG, 1.6 kb SalI–HindIII fragment of pCAC1; coxI, 1.0 kb EcoRV–HindIII of pCAC9; coxK, 1.3 kb HindIII–EcoRV of pCAC9; cbbR, 1.5 kb EcoRV–SalI fragment of pBS7.6; cbbLS, 1.4 kb EcoRI fragment from pBS5.7; hoxL, 0.85 kb fragment of pBS19; hoxS, 2.2 kb EcoRV fragment of pBS2.2.

B. Santiago et al. / Gene 236 (1999) 115–124

pBluescript yielding the plasmids pCAC1 and pCAC9 (Fig. 1). Deletion derivatives of the plasmids were generated and both strands were sequenced. To position nonoverlapping fragments a single strand of pKT7-9 and pBS7.6 was also sequenced (Fig. 1). The nucleotide sequence upstream of coxMSL (3815 bp) comprises 3999 nt and includes the open reading frames designated orf7, coxB and coxC which are arranged in the transciptional order 5∞ orf7coxB coxC 3∞ (Fig. 1). The corresponding ribosomal binding sites could be identified on the nucleotide sequence, since they are complementary to the 3∞ terminus of the 16s rRNA of O. carboxidovorans (Auling et al., 1988). Plausible −10/−35 promotor sequences appear 180 nt upstream of coxB, 70 nt upstream of coxC and 58 nt upstream of coxM. A possible terminator sequence is located 16 nt downstream of coxB. The sequence downstream of coxL comprises 8084 nt and includes the open reading frames designated coxD, coxE, coxF, coxG, coxH, coxI and coxK ( Fig. 1). Six of these are arranged in the transciptional order 5∞ coxDcoxEcoxFcoxGcoxHcoxI 3∞, whereas coxK is transcribed in the opposite direction. CoxD has been sequenced and characterized before as orf4 (Schu¨bel et al., 1995). The ribosomal binding sites of the remaining genes could be identified on the nucleotide sequence. A plausible −10/−35 promotor sequence could be identified 84 nt upstream of coxH. The codon usage of the newly identified genes and the structural cox and hox genes is similar (Schu¨bel et al., 1995; Santiago and Meyer, 1997). 3.2. Sequence comparison and characteristics of the identified orfs The main characteristics of the identified cox genes are summarized in Table 2. In addition, the following similarities were found between the Cox polypeptides and heterologous proteins: Orf7 and CoxB showed no significant sequence homologies to other proteins in database comparisons. CoxC is surprisingly similar to the CoxH polypeptide from O. carboxidovorans (42% identity, 79% similarity). CoxC is also similar to a sensory histidine kinase (35% identity, 65% similarity in aa 1–186 of CoxC ) from Synechocystis ( Kaneko et al., 1996) and to the YKOW protein (29% identity, 67% similarity in aa 50–232 of CoxC ) of Bacillus subtilis (Ogasawara et al., 1994). CoxC shows also 43% identity (79% similarity) in aa 300–402 to the translated region (nt 1–409) upstream of cutM (nt 519–1382) from H. pseudoflava ( Kang and Kim, 1999). CoxD shares the following sequence similarities: 50% identity (85% similarity) to an Orf4 protein of Bradyrhizobium japonicum (Lorite et al., 1997), 48% identity (72% similarity) to the deduced protein MTV036.05c of Mycobacterium tuberculosis (Philipp

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et al., 1996), and 44% identity (79% similarity) to an Orf1 protein of Rhodococcus sp. (Nagy et al., 1995). CoxE is similar to the deduced Orf5 protein (33% identity, 73% similarity) of B. japonicum (Lorite et al., 1997), and to the deduced protein MTV036.03c (30% identity, 60% similarity) of M. tuberculosis (Philipp et al., 1996). CoxF shows 31% identity and 58% similarity to the deduced protein MTV036.07c (264 aa) of M. tuberculosis (Philipp et al., 1996). In addition, the N-terminus of CoxF (aa 1–100) shows 31% identity (63% similarity) to the deduced Orf6 protein (107 aa) of B. japonicum (Lorite et al., 1997). The aa 125–275 of CoxF show 29% identity (59% similarity) to the deduced Orf7 protein (176 aa) of B. japonicum (Lorite et al., 1997). CoxG shows 59% identity (94% similarity) to a deduced polypeptide upstream of CoxM in B. japonicum (Lorite et al., 1997). CoxG is also similar to the deduced 171 aa protein MTV036.04c (30% identity, 63% similarity in aa 30–155 of CoxG) of M. tuberculosis (Philipp et al., 1996) and to an 171 aa Orf4 protein (27% identity, 66% similarity in aa 30–141 of CoxG) of P. thermocarboxydovorans (Pearson et al., 1994). CoxH is similar to the CoxC polypeptide of O. carboxidovorans (42% identity, 79% similarity). It is also similar to a sensory histidine kinase (29% identity, 61% similarity in aa 1–320 of CoxH ) from Synechocystis ( Kaneko et al., 1996) and to the YKOW protein (31% identity, 67% similarity in aa 50–231 of CoxH ) of B. subtilis (Ogasawara et al., 1994). CoxH of O. carboxidovorans contains the sequence 57MHFIGI–X – 58 MHYLGM–X –MHYTAM188 which is also present in 56 CoxC of O. carboxidovorans (57MHFVGM–X – 55 MHYIGM–X –MHYTAM185), the histidine kinase of 56 Synechocystis (73MHFIGM–X –MHYSGM–X – 58 61 MHYTAM209), and the identified protein YKOW of B. subtilis (8MHFVGI–X –MHYIGM–X –MHYTG56 61 M144). CoxH shows also significant similarities to different permeases, which are the 448 aa gluconat permease (24% identity, 56% similarity in aa 45–281 of CoxH ) of B. subtilis ( Fujita et al., 1986) and the 419 aa cytosine permease (26% identity, 56% similarity in aa 2–217 of CoxH ) of E. coli (Danielsen et al., 1992). CoxI is similar (27% identity, 60% similarity) to the deduced protein MTV036.11c (Philipp et al., 1996) of M. tuberculosis, and to the deduced protein YagQ (32% identity, 62% similarity in aa 43–303 of CoxI ) of E. coli (Blattner et al., 1997). In addition, aa 149–306 of CoxI show 28% identity (67% similarity) to aa 122–272 of CoxF of O. carboxidovorans. CoxK shows 25% identity and 60% similarity to the hypothetical protein YYAM of B. subtilis (Ogasawara et al., 1994). 3.3. CO-specific transcription In O. carboxidovorans growing chemolithoautotrophically with CO, all the genes assembled from coxB to

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Table 2 Characteristics of the identified genes and the deduced polypeptides Gene/gene product

Nucleotides/ amino acids

Predicted molecular mass (Da)

Motifs or special features

Potential transmembranous helices (aa)

orf7/Orf7 coxB/CoxB coxC/CoxC

336/112 1005/335 1206/402

12 141 35 437 42 862

none none none

coxM/CoxM

864/288

30 239

coxS/CoxS

498/166

17 792

coxL/CoxL

2427/809

88 735

coxD/CoxD coxE/CoxE coxF/CoxF

885/295 1200/400 843/281

33 367 44 232 29 372

coxG/CoxG coxH/CoxH

618/206 1203/401

21 558 42 517

FAD pyrophosphate binding: 32AGGHS36, 111TIGG114, 193Y type I 2Fe:2S: 102CX CX CNC139 2 31 type II 2Fe:2S: 42CX CX CX C62 4 2 11 388S-selanylcysteine loop: 384VAYRCSFR391 MCD contacting segments: 240Q, 270G, 528QGQGHETY535, 569GSRST573, 586CGTRIN591, 761VGE763 Mononucleotide binding site: 43GEAGVGKT50 12.5% arginine Histidine acid phosphatases phosphohistidine signature: 258VAEMVEIRRHGQRQS272 none none

none 31–47 41–57, 79–95, 103–119, 143–159, 164–180, 208–224 none

coxI/CoxI

996/332

36 681

coxK/CoxK

888/296

30 994

9.6% arginine Mononucleotide binding site: 276GLYFGGKS283 none

cbbS are specifically transcribed which is documented in Fig. 2 by a selected number of representative slot–blots. These genes are not transcribed under heterotrophic conditions ( Fig. 2). The data indicate a coordinate transcription of the coxMSL structural genes along with the cbb genes, and the accessory genes coxB, coxC and coxD to coxK (Fig. 2). Under heterotrophic conditions in the presence of CO, cox gene transcription was only 5–10%, and the cbb genes were not transcribed at all, indicating that the gene region extending from coxB to cbbS is inducible by CO, but also subject to repression by organic substrates. HoxS, hoxL, cbbR, cbbL and cbbS were specifically transcribed in O. carboxidovorans growing chemolithoautotrophically with H plus CO 2 2 (Fig. 2), referring to a coordinate regulation of the hox genes and the cbb genes. The absence of cox gene specific mRNA under these conditions indicates that cox gene transcription has a specific requirement for CO, and that chemolithoautotrophic conditions alone are not sufficient. The genes extending from coxB to coxK are clustered and subject to CO-specific coordinate transcription, and therefore designated as the cox gene cluster. 3.4. Mutants of O. carboxidovorans defective in the utilization of CO We were interested to identify genes functional in the chemolithoautotrophic utilization of CO on the plasmid

none none

none none none none 49–65, 79–95, 106–122, 146–162, 212–228 none 48–64, 78–94, 109–125, 133–149, 164–180, 191–207, 224–240, 251–267, 277–293

pHCG3. For transposon mutagenesis the Tn5containing plasmid pSUP1011 was transferred from E. coli S17-1 to O. carboxidovorans. Transconjugants occurred at a frequency of 4.5×10−6 per donor. A total of 612 kanamycin-resistant transconjugants were obtained. Of these, 21 were impaired in the chemolithoautotrophic utilization of CO (phenotype CO−). 18 of the CO− mutants retained the ability to utilize H 2 plus CO (phenotype CO−H+). The phenotype 2 2 CO−H+ indicates mutations in genes essential for the 2 utilization of CO and excludes mutations in cbb genes. Three transconjugants were CO−H−, suggesting mut2 ations in cbb genes. Three other transconjugants were CO+H−, referring to mutations in hox genes. 2 Southern hybridizations of total DNA from the transconjugants with the phenotype CO−H+ revealed that 2 the mutants TN-81, TN-106, TN-398, TN-424 and TN-470 of O. carboxidovorans carry the transposon on the plasmid pHCG3. Hybridizations of restriction fragments indicated that the transposon had been integrated either into the cox gene cluster [mutants TN-81, TN-106 ( Fig. 1)] or into genes outside the entire 30 kb cox–cbb– hox chemolithoautotrophy region [mutants TN-398, TN-424, TN-470 ( Fig. 1)]. A 11.4 kb hybridizing EcoRV fragment of TN-81 and a 8.6 kb EcoRV fragment of TN-106 were subcloned into pBluescript KS, yielding the plasmids pTn81 and pTn106, respectively. Sequencing revealed that coxH

B. Santiago et al. / Gene 236 (1999) 115–124

was interrupted by the transposon in the mutant TN-81 (Fig. 1). With the mutant TN-106 the transposon is located in the intergenic region between coxH and coxI, suggesting that the mutation originates from a polar effect of the transposon on coxI ( Fig. 1). Although the mutants TN-81 and TN-106 were not able to grow with CO, they both could synthesize CO dehydrogenase when growing with organic substrates in the presence of CO. Under these conditions CO dehydrogenase appeared with similar specific activity of about 26 nmol CO oxidized min−1 mg protein−1 in crude extracts of the mutants and wild type bacteria. The wild type and mutant enzymes had similar mobilities upon native PAGE, and SDS-PAGE revealed an indistinguishable subunit composition. The data show that the gene products of coxH and coxI are not involved in the synthesis of catalytically competent CO dehydrogenase. The sequences of CoxH and CoxI indicate that both are not a cytochrome. CoxH is predicted as a membrane protein, and may be one of the proteins engaged in the interactions of CO dehydrogenase with the cytoplasmic membrane. Since CoxI is predicted as a cytoplasmic protein it is difficult to imagine any specific functions in CO metabolism right now. A 9.5 kb hybridizing ClaI fragment of TN-398 was cloned, yielding the plasmid pTn398, and sequenced. The sequence revealed the 3∞ terminus of an orf, which gene product (200 aa) showed high similarities (60–68% identity, 84–90% similarity) to the deduced polypeptide of clpA of different bacteria. Clp proteases consist of two different components [ClpA (84 kDa) and ClpP (21 kDa)], which both are required for proteolysis (Goldberg et al., 1994). The transposon was identified 530 nt from the 3∞ terminus of a second orf (2370 nt), which deduced protein (790 aa, 87.546 kDa) shows high similarities (63–67% identity, 78–84% similarity) to Lon from different bacteria. The lon gene of E. coli and other organisms codes for an ATP-dependent and DNAbinding protease which plays multiple regulatory roles including proteolytic degradation of several classes of regulatory and abnormal proteins, radiation resistance, cell division, filamentation, and production of capsule polysaccharide (Goldberg et al., 1994). In E. coli, lon mutations have pleiotropic consequences, giving rise to filamentous growth, overproduction of capsular polysaccharide, and increased sensitivity to DNA-damaging agents (Gottesmann and Maurizi, 1992). In contrast, the lon insertional mutant O. carboxidovorans TN-398 was specifically impaired in the chemolithoautotrophic utilization of CO, whereas chemolithoautotrophic growth with H plus CO or heterotrophic growth with 2 2 pyruvate were not affected. In addition, the lon mutants exhibited no filamentation or increased polysaccharide production. The mutant O. carboxidovorans TN-398, which provides the second example of a lon mutation exhibiting clear phenotypic consequences, can be under-

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stood in analogy to the lon::cat null mutant of B. subtilis (Schmidt et al., 1994). In B. subtilis, Lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sG, and sG is possibly a direct substrate for degradation by Lon protease (Schmidt et al., 1994). How could the absence of Lon protease produce the phenotype CO− in the lon mutant of O. carboxidovorans? As discussed above, the cox gene cluster is subject to induction by CO and repression by organic substrates. Degradation of the repressor protein by Lon protease would result in derepression of the cox genes, which cannot take place in the lon mutant of O. carboxidovorans. A 8.7 kb hybridizing ClaI fragment of TN-424 was cloned, yielding the plasmid pTn424, and sequenced. The transposon was identified 108 nt away from the 3∞ terminus of an orf ( Fig. 1), which deduced polypeptide (35 aa) showed 63% identity and 80% similarity to CycH from B. japonicum. A second orf was identified 60 nt downstream of cycH, which deduced polypeptide (166 aa, 18 017 Da) showed 50–69% identity (63–86% similarity) to CycJ from different bacteria. cycJ and the orf downstream of cycJ overlap in 4 nt. From this orf, 600 nt were sequenced and the deduced poypeptide (200 aa) showed 66–84% identity (76–92% similarity) to the N-terminus of CycK (660–680 aa) from different bacteria. The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in different bacteria, and has been suspected to be involved in the attachment of the heme to apocytochrome c (Ritz et al., 1995; Delgado et al., 1995). In O. carboxidovorans the cycHJK gene products might have similar functions in the biosynthesis of a cytochrome specifically required in CO metabolism. A 8.6 kb hybridizing EcoRI fragment of TN-470 was cloned, yielding the plasmid pTN470, and sequenced. The transposon was identified in a gene region (orfX ), which deduced gene products show no motifs and exhibit no significant sequence homologies in database searches.

4. Discussion The sequence, transcriptional, mutational and physiological analyses presented here suggest that the CO dehydrogenase of O. carboxidovorans is an integral and unique part of an elaborate CO oxidizing system which is encoded by cox structural genes and cox accessory genes that reside on the megaplasmid pHCG3 (Fig. 1). The cox structural genes along with nine accessory genes are assembled in the 14.5 kb cox gene cluster (Figs. 1 and 2) and are transcribed in a CO-dependent manner ( Fig. 2). In addition to the accessory cox genes, mutational analyses have identified the genes lonA, cycH and orfX with essential functions in the utilization of CO

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Fig. 3. Organization of the cox and cox like genes in different bacteria. Black shadows represent the CODH structural genes. Same shading refers to similar genes. White arrows represent genes without homologies.

(Fig. 1). These genes reside outside the 30 kb cox–cbb– hox chemolithoautotrophy region on pHCG3. Primary sequence analysis in databanks did not propose any functions for the products of the accessory cox genes, which emphasizes their specific role in CO metabolism. Nevertheless, it is apparent that the cox gene cluster encodes a striking number of deduced membrane proteins containing one (CoxB), five (CoxH ), six (CoxC ) or even nine (CoxK ) transmembranous helices. Biochemical work indicates that CoxD, which is predicted as a soluble protein, in fact also is an integral membrane protein ( Wengert et al., personal communication). These proteins neither look like conventional electron transfer components, such as cytochromes or iron–sulfur proteins, nor like gene products involved in the biosynthesis of the molybdopterin cofactor (MoaA, MoeA, MoaC ) or the high affinity transport of molybdate (ModA, ModB, ModC ). Nicotine dehydrogenase of Arthrobacter nicotinovorans is another member of the molybdenum hydroxylase sequence family, and the moa, moe and mod genes are grouped together with the clustered ndhABC structural genes on the 160 kb plasmid pAO1 (Menendez et al., 1997). CO dehydrogenase in intact cells of O. carboxidovorans is associated with the inner aspect of the cytoplasmic membrane (Meyer et al., 1990). The enzyme binds in a non-covalent fashion and can be removed from membranes by solubilization with the denaturing non-ionic detergent dodecyl b-D-maltoside (Meyer et al., 1990). In vitro reconstitution of depleted membranes requires cations and is specific for CO-grown bacteria. It is, therefore, tempting to assume that one or several of the membrane proteins specifically induced by CO are

involved in anchoring CO dehydrogenase to the cytoplasmic membrane. Binding of CO dehydrogenase to its membrane anchor is related to the physiological state of the bacteria (Meyer et al., 1990). The CoxD membrane protein would be able to act in signal transduction via its predicted mononucleotide binding site. CO dehydrogenases have been biochemically characterized and the structural genes have been sequenced from O. carboxidovorans, P. thermocarboxydovorans and H. pseudoflava (Fig. 3). Database searches reveal genes in B. japonicum and M. tuberculosis which contain motifs and sequences also specific for CO dehydrogenases ( Fig. 3). With the exception of B. japonicum, in all these bacteria the CO dehydrogenase structural genes are clustered in the transcriptional order 5∞ coxM (cutB, cutM, MTV036.10c)coxS (cutC, cutS, MTV036.9c) coxL (cutA, cutL, MTV036.8c) 3∞. The coxM like genes in the different bacteria encode a flavoprotein equivalent to CoxM of O. carboxidovorans, as is apparent from high overall sequence similarities (30–50% identity, 59– 71% similarity). In addition, the structurally identified double glycine motifs (Dobbek et al., 1999) involved in binding the FAD pyrophosphate in CoxM of O. carboxidovorans are present [CoxM (Oc), CutB, CutM: 32MAGGHS; CoxM (Bj), MTV36.10c: 19MAGGHS and CoxM (Oc), CutB, CutM: 111MTIGG; CoxM (Bj): 108MTIGG; MTV36.10c: 103MTLGG ], and the aromatic residue shielding the center of the isoalloxazine ring [CoxM (Oc): 193MY; CutB, CutM: 193MW; CoxM (Bj) 183MY; MTV36.10c: 184MW ] is also present. The coxS like genes in the different bacteria encode an iron–sulfur protein equivalent to CoxS of O. carboxidovorans. This is indicated by high overall sequence

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similarities (57–64% identity, 72–81% similarity) and motifs characteristic of 2Fe:2S clusters of the N-terminal type II (CX CX CX C ) and the C-terminal type I 4 2 11 (CX CX CNC ) which have been structurally charac2 n terized in the CO dehydrogenase from O. carboxidovorans (Dobbek et al., 1999). The coxL like genes in the different bacteria encode an equivalent to CoxL of O. carboxidovorans, as is indicated by high overall sequence similarities (57–67% identity, 78–85% similarity) and a catalytic site with a unique conserved loop [CoxL: 384LVAYRCSFR, CutC: 378LVAYRCSFR, CutL: 381LVAYRCSFR; MTV36.8c: 377LVAYACSFR] carrying in O. carboxidovorans the residue S-selanylcysteine 388L, a conserved Glu [CoxL: 240LQ; CutC: 234LQ; CutL: 237LQ; MTV36.8c: 233LQ ] and a conserved Gln [CoxL: 763LE; CutC: 754LE; CutL: 757LE; MTV36.8c: 752LE]. With the exception of CoxB and CoxK, all deduced products of the genes of the cox gene cluster of O. carboxidovorans have counterparts in the corresponding gene clusters of the bacteria depicted in Fig. 3. Besides, all deduced accessory Cox polypeptides were not significantly similar to any other proteins in the databases. It has been discussed that genes in a particular cluster usually encode proteins that catalyze mechanistically diverse reactions which together serve a common function or contribute to a phenotype (Roth et al., 1996). The prominent clustering of the genes coxMSL along with coxDEFG in O. carboxidovorans, which is also apparent in M. tuberculosis, B. japonicum and H. pseudoflava ( Fig. 3), refers to a functional relation of the coxDEFG gene products and CO dehydrogenase. In the different systems the genes equivalent to coxDEFG are grouped together, although their individual order might differ, and are arranged immediately downstream of the CO dehydrogenase structural genes (Fig. 3). The only modifications are that in B. japonicum the gene equivalent to coxG precedes the coxMS structural genes and that in M. tuberculosis the additional gene MTV036.06c is inserted. The cox genes encode components required for the chemolithoautotrophic utilization of CO, which for most organisms is a noxious compound. Their clustering in O. carboxidovorans and the other bacteria discussed further substantiates the view that genes encoding pathways for the utilization of relatively rare carbon sources are virtually all clustered in bacterial genomes ( Roth et al., 1996). The phototrophic purple bacterium Rhodospirillum rubrum can grow with CO as the sole source of energy during anaerobic growth in the dark employing a Ni– CO dehydrogenase ( Kerby et al., 1992) which is unrelated to the structurally characterized Mo–CO dehydrogenase of O. carboxidovorans. Like the cox genes in O. carboxidovorans, the CO dehydrogenase genes cooFSCTJA of R. rubrum are clustered, and the tran-

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scription of cooFSCTJ is induced by CO ( Kerby et al., 1997). The cooF gene encodes a ferredoxin like protein ( Kerby et al., 1992), cooS encodes the Ni-containing CO dehydrogenase ( Kerby et al., 1992), the cooCTJ region is involved in Ni insertion ( Kerby et al., 1997) and cooA codes for a CO responsive transcriptional activator (Shelver et al., 1995). Besides CO sensing, all of these functions and genes are specific for Ni–CO dehydrogenase. Accordingly, the cox gene cluster of O. carboxidovorans is not related to the coo gene cluster of R. rubrum, and both clusters do not have any genes in common.

Acknowledgements We thank Ms. Elisabeth Keese for expert technical assistance and Professor A. Pu¨hler (Bielefeld, Germany) for providing the plasmid pSUP1011. This work was supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany), the Fonds der Chemischen Industrie ( Frankfurt a.M., Germany), and the Bayerisches Staatsministerium fu¨r Landesentwicklung und Umweltschutz (Mu¨nchen, Germany).

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