INTRODUCTION

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The neutrophilic facultatively lithoautotrophic gram-negative bacterium Paracoccus pantotrophus grows aerobically with a large variety of carbon sources and with molecular hydrogen or thiosulfate as energy source, and nitrate serves as electron acceptor under anaerobic conditions (41). P. pantotrophus (formerly Paracoccus denitrificans and Thiosphaera pantotropha [31, 38, 41]) has a branched electron transfer network. The electron flux is regulated by the reduction of the Q pool (33). While the transfer of electrons by the terminal cytochrome oxidases aa₃ and cbb₃ results in a translocation of six protons (6H⁺/2e) (36, 54), the reduction of a terminal electron acceptor by the quinol oxidase o results in a translocation of only four protons (4H⁺/2e) (39).

To identify the components involved in energy transformation by oxidation of reduced inorganic sulfur compounds (Sox) of P. pantotrophus GB17, genetic studies were initiated by the isolation of mutants. Transpositional mutagenesis with Tn5-mob coding for kanamycin resistance yielded mutants defective in Sox. One mutant, TP43, is unable to grow with thiosulfate (Sox) or reduce nitrite under anaerobic conditions (Nitd), while reduction of nitrate to nitrite is not affected (11).

Cytochromes of the c type are distinguished from cytochromes of other classes by the covalent attachment of their prosthetic heme group to the conserved CXXCH motif of the apoprotein. The biogenesis of c-type cytochromes consists of several steps performed in the cytoplasm and in the periplasm (reviewed in reference 53). Two different systems with respect to the late steps of cytochrome c biogenesis have been described for bacteria (reviewed in reference 28). Most gram-positive bacteria possess the class II system, consisting of at least 4 proteins (ResA, CcsA, CcsI, and CcdA), while most gram-negative bacteria harbor the class I system, consisting of at least 11 proteins (DsbABD and CcmABCDEFGH). In gram-negative bacteria the addition of the heme group to the apoprotein takes place in the oxidative environment of the periplasm, where disulfide bonds between pairs of protein cysteine thiols are generated by the DsbA/B system (4). Thus, cytochrome c apoproteins have to be rereduced prior to heme binding. Recently three proteins, CcmH, CcmG, and DsbD, were proposed to be responsible for this step in cytochrome c maturation in Escherichia coli (15). According to this proposal, the oxidized cytochrome c apoprotein is reduced by CcmH and CcmG and the latter is rereduced by DsbD. In Rhodobacter capsulatus, CcdA has been described as homologous to DsbD of E. coli, having similar predicted structural characteristics and essential cysteine residues (14). In P. pantotrophus within the sox gene cluster coding for lithotrophic sulfur oxidation, ORF2 has been identified, with a predicted product that is about 35% identical to CcdA of Bacillus subtilis and about 45% identical to CcdA of R. capsulatus. Eight genes required for cytochrome c biogenesis including ccmG have been identified so far in P. denitrificans. CcmG of P. denitrificans was proposed to reduce disulfide bonds in vivo (34) and may have the same function in cytochrome c biogenesis as that proposed for CcmG of E. coli.

To determine the nature of the mutation in the Sox mutant TP43, we have reexamined its pleiotropic character and have identified the complete absence of c-type cytochromes in this strain. We have cloned the Tn5-mob-containing DNA region of strain TP43 (11) and have isolated and sequenced the corresponding wild-type gene region. Below we describe a new gene, ccdA, of P. pantotrophus GB17 which is essential for cytochrome c biogenesis and which is suggested to be involved in the transfer of reducing equivalents from the cytoplasm to the periplasm.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. Strains and plasmids used and constructed in this study are listed in Table 1.

Media and growth conditions. Mineral media were identical for heterotrophic and lithotrophic growth of P. pantotrophus and were described previously (11). P. pantotrophus was cultivated at 30°C. For mixotrophic growth with thiosulfate, mineral media contained 20 mM sodium succinate and 20 mM sodium thiosulfate at an initial pH of 8. For anaerobic growth, bacteria were cultivated in Luria-Bertani broth containing 0.1% (wt/vol) sodium nitrite or 0.1% (wt/vol) potassium nitrate. The following antibiotics were used when appropriate for P. pantotrophus GB17: kanamycin (KM) at 300 µg/ml, tetracycline (TC) at 5 µg/ml, and chloramphenicol (CM) at 5 µg/ml. For E. coli the antibiotics were KM at 50 µg/ml, ampicillin (AP) at 50 µg/ml, TC at 12.5 µg/ml, and CM at 30 µg/ml. Antimycin A was used at a concentration of 50 µg/ml. Cellular yields of P. pantotrophus were determined as described for Ralstonia eutropha (19).

Enzyme assays. Whole cells were used for enzyme assays. Thiosulfate oxidizing activity was determined with an oxygen electrode as described elsewhere (56). N, N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) oxidase activity was measured in an oxygen electrode in the presence of 1 mM TMPD. One unit of enzyme activity was defined as 1 µmol of substrate converted per min at 30°C.

Immunoblot analysis. The level of Sox-specific c-type cytochrome SoxD and the purity of periplasmic extracts were determined by immunoblot analysis as described elsewhere (56). Antibodies against SoxD antigens were obtained against the oligopeptide FYPDDRDQTEYPLF, deduced from the soxD nucleotide sequence. Antibodies were raised in rabbits at the facilities of Eurogentec (Seraing, Belgium). Antibodies against ribulose-bisphosphate carboxylase of R. eutropha were obtained from B. Bowien, Goettingen, Germany (7).

Cytochrome analysis. Cytochromes of P. pantotrophus GB17 and its derivative strain TP43 were analyzed by using cell extracts. Cells were cultivated as specified below, washed twice at 0°C, and resuspended in 10 mM HEPES buffer, pH 7.4. Cells were disrupted by French press, and cell extracts were obtained from the 30,000 × g supernatant (16). For analysis of c-type cytochromes from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were prepared without boiling. Cytochromes were stained as described by Francis and Becker (18). For identification of the cytochromes, cell extracts (10 mg of protein/ml) were subjected to redox difference spectroscopy. Cell extracts reduced by addition of solid sodium dithionite were measured at 20°C against air-oxidized extracts with a Shimadzu UV 160 A spectrophotometer. Cytochromes were quantified from the 30,000 × g supernatant by the pyridine extraction procedure (5).

Analytical procedures. Formate, thiosulfate, and nitrite were quantified by colorimetric methods. Formate was measured by the procedure described by Lang and Lang (30), thiosulfate was measured by the procedure described by Sørbø (49), and nitrite was measured by the sulfanilamide method (23). Protein from cell extracts was quantified by the procedure described by Bradford (8).

DNA and RNA techniques. Standard DNA techniques (43) were used. Plasmid DNA was isolated by the procedure described by Birnboim and Doly (6) or by using the high pure-plasmid isolation kit (Boehringer, Mannheim, Germany) according to the manufacturer's protocol. Total RNA was isolated as described elsewhere (24) or by using the high pure-RNA isolation kit of Boehringer. DNA sequencing was performed by primer walking with the thermostable DNA polymerase of Thermus aquaticus and 7-deaza-dGTP (Amersham-Buchler, Braunschweig, Germany) by the dideoxy-chain termination method (44) with an automated DNA sequencing system (Li-Cor; MWG-Biotech, Munich, Germany). The plasmids pBSKXP6.8 and pBSKB3.8, used for sequencing, are described in Table 1.

Construction of pEF15. The Tn5-mob-containing EcoRI fragment of strain TP43 was cloned in pSUP205 at the facilities of Biodelta (Bad Oeynhausen, Germany). Total DNA of strain TP43 was digested with EcoRI. Fragments of 18 to 6 kb were separated by agarose gel electrophoresis and purified with the extraction kit Jetsorb (Genomed, Bad Oeynhausen, Germany). Fragments were ligated with pSUP205, and the resulting plasmids were transformed into E. coli XL1-Blue. Transformants exhibiting the Tc^r, Km^r, Ap^r, and Cm^S phenotypes were examined for hybrid plasmids. One clone contained the 18-kb hybrid plasmid pE18TP43. The insertion of Tn5-mob was verified by Southern hybridization, and its location was determined by physical mapping as well as by DNA sequencing (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Physical map of the insert DNAs of the plasmids pE18TP43 and pEF15 and ORFs within the 2.8-kb ccdA gene region. E, EcoRI; S, SmaI; P, PstI; C, ClaI; B, BamHI; N, NotI.

Construction of pVK1 to complement ccdA::Tn5 in TP43. Primer C1 (5'-ATGTTGGGAATCGAGCTTGCA-3') and primer C2 (5'-CTAACCCAGCGTGGCCAGCCA-3') were used to amplify the ccdA gene region by PCR (32). The resulting PCR product was cloned into the EcoRV site of pBSK to produce pBSKccdA. The insert DNA was then isolated by EcoRI and HindIII and cloned into the plasmid pVK101. The resulting plasmid pVK1 was transformed into E. coli S17-1 and conjugated therefrom into the mutant strain TP43 to produce P. pantotrophus TP43(pVK1).

Nucleotide sequence accession number. The nucleotide sequence reported here has been deposited in the EMBL and GenBank databases under accession number AF308446.

	RESULTS

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Physiological characterization of strain TP43. Strain TP43 is unable to grow lithotrophically with thiosulfate and unable to reduce nitrite anaerobically (11). The pleiotropic character may have resulted from a mutation in a regulatory mechanism or may have been related to the energy metabolism. To distinguish between these possibilities, the cellular yields were determined. When cultivated with glucose, glyoxylate, or formate, respectively, strain TP43 exhibited generally lower cellular yields of 11.3 to 20.7% compared to the wild type (Table 2). The reduced yield was evidence that the mutation (i) was not restricted to the phenotypes described initially (11) but was a general characteristic of strain TP43 and (ii) was linked to the energy metabolism. Therefore, the effect of antimycin A, an inhibitor of the electron transfer to the bc₁ complex of the respiratory chain in various bacteria (reviewed in reference 40), was examined under anaerobic growth conditions with nitrate as electron acceptor. These conditions allowed the determination of whether c-type or other types of cytochromes were affected. Nitrate reduction does not require c-type cytochromes in P. denitrificans, whereas nitrite reduction involves different c-type cytochromes linked to the bc₁ complex, nitrite reductase, nitric oxide, and nitrous oxide reductase (10, 52). Under anaerobic conditions in the presence of antimycin A the yield of the wild type was reduced by 55% and the specific growth rate was reduced from 0.38 to 0.25 per h. The extent and the rate of growth of strain TP43 without antimycin A were identical to those of the wild type cultivated with antimycin A, and addition of the drug did not affect the growth characteristics of TP43 (data not shown). Under anaerobic growth conditions addition of antimycin A to the wild type allowed quantitative reduction of nitrate; however, nitrite was not metabolized further (data not shown). These results pointed to the inability of strain TP43 to form a component essential for the cytochrome bc₁ complex or the electron flow via this complex. This conclusion was supported by the complete absence of TMPD oxidase activity in strain TP43 (Table 2). For oxidation of TMPD, c-type cytochromes are essential (26).

Cytochrome c analysis. To analyze components involved in electron transport, cytochromes were analyzed for P. pantotrophus GB17 and TP43. Redox difference spectroscopy of extracts from glucose-grown cells of P. pantotrophus GB17 showed an absorption maximum at 551 nm, diagnostic of c-type cytochromes, while extracts from glucose-grown cells of TP43 showed an absorption maximum at 560 nm, typical of b-type cytochromes (Fig. 2). Quantitative analysis of pyrimidine hemochromes (5) from the wild type and strain TP43 revealed a maximum concentration of 1.83 µmol of cytochrome c per g of protein for the wild type, while only traces (0.09 µmol of cytochrome c per g of protein) were detected from strain TP43. Using this method, the concentrations of b-type cytochromes (5) from the wild type and strain TP43 were identical (0.3 µmol per g of protein; data not shown). The c-type cytochromes observed for strain TP43 were less than 5% of those for the wild type. This amount was attributed to the method of detection and its interference with the spectrometric determination of b- and a-type cytochromes as previously discussed (5).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Visible absorption spectra of total soluble cell extracts from P. pantotrophus GB17, strain TP43, and strain TP43(pVK1). The cuvettes contained 10 mg of protein per ml. The samples were reduced by addition of solid sodium dithionite, and spectra were measured against air-oxidized references.

View larger version (101K): [in this window] [in a new window]   FIG. 3.   Heme staining of total soluble extracts from P. pantotrophus GB17, strain TP43, and TP43(pVK1). Samples of cell extract of each strain (0.3 mg of protein) were subjected to SDS-PAGE without boiling.

Cloning and physical map of the ccdA gene region. The wild-type gene region of 15 kb was cloned as described in Materials and Methods and was physically mapped. The map was compared with the Tn5-mob-containing insert cloned in pE18TP43. Identical restriction sites were obtained within the 12.5-kb EcoRI fragments of the two plasmids. The position of Tn5-mob mapped between the EcoRI and BamHI sites of pE18TP43. The precise position of the Tn5-mob inverted-repeat chromosome junction was determined by sequencing to be between nucleotides 1697 and 1698 (Fig. 1).

Sequence analysis The nucleotide sequence of 2.8 kb was determined for both strands starting from the EcoRI restriction site. The sequence was consistent with the restriction enzyme cleavage map of pEF15. Analysis of this sequence revealed three open reading frames (ORFs) designated ORFA, ccdA, and ORFB. These ORFs revealed coding characteristics according to codon preference analysis (50). ORFA was separated from ccdA by 48 nucleotides. Within this stretch was located an inverted repeat of 23 nucleotides with the potential to form a hairpin structure with a free energy of formation of 100.4 kJ/mol. ORFB was separated from ccdA by 9 bp and transcribed in the opposite direction.

Complementation of the mutation in strain TP43. To analyze if the insertion of the transposon Tn5-mob in ccdA caused any polar effects which could have been responsible for the pleiotropy of TP43, plasmid pVK1 was constructed. The ccdA gene region was cloned into the vector pVK101 and used for complementation. Strain TP43 harboring plasmid pVK1 in trans was able to grow aerobically with thiosulfate and anaerobically with nitrite (data not shown). When cultivated heterotrophically with glucose, glyoxylate, or formate, the cellular yields of TP43(pVK1) were similar to those of the wild type (Table 2). Spectrophotometric analysis demonstrated that the ability to form holo- c-type cytochromes was fully restored (Fig. 2), as was also evident from heme staining (Fig. 3). These data were convincing evidence that the pleiotropy of strain TP43 resulted from the inactivation of ccdA.

Transcription of ORFA and ccdA. Transcription of ORFA started with an adenine 114 nucleotides before the translation start codon as determined by primer extension analysis using the oligonucleotides PE3 and PE4 (data not shown). Using the oligonucleotides PE1 and PE2, complementary to the beginning of the subsequent ccdA gene, the signal appeared at sizes of about 1,000 and 1,200 nucleotides, respectively (data not shown). This result was evidence that ORFA and ccdA were cotranscribed. Constitutive expression of ORFA and ccdA was deduced from primer extension analysis using oligonucleotide PE3. Signals were equally intense in cells grown aerobically with glucose or succinate and cells grown anaerobically with nitrate as electron acceptor (data not shown).

Biochemical analysis of the ccdA function. To determine the function of ccdA expression of SoxD, a c-type cytochrome was monitored. The c-type cytochrome SoxD is associated with a sulfite dehydrogenase homologue, located in the periplasm and beneficial for thiosulfate oxidation (37, 56). SoxD antigens were formed by strain TP43 when cultivated in the presence of thiosulfate, demonstrating that expression of the sox genes was not affected by the mutation. Moreover, SoxD antigens were detected in the periplasm using the chloroform procedure which specifically extracts proteins therefrom (Fig. 4). This result demonstrated that the ccdA gene was not involved in the transport of the apoprotein SoxD to the periplasm but was involved in a late step of cytochrome c maturation.

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Western blot analysis after SDS-PAGE of cell extracts of P. pantotrophus GB17 and strain TP43 for ribulose-bisphosphate carboxylase (A) and cytochrome SoxD antigens (B). (A) Proteins (15 µg) of the cytoplasmic and periplasmic fractions of strain GB17 (lanes 1 and 3, respectively) and strain TP43 (lanes 2 and 4, respectively). (B) homogenous SoxD (2 µg; lane 1), periplasmic fraction of strain GB17 (10 µg of protein; lane 2), and periplasmic fraction of strain TP43 (20 µg of protein; lane 3).

	DISCUSSION

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Three new ORFs, ORFA, ccdA, and ORFB, were identified for the genome of P. pantotrophus GB17. ORFA and ccdA were constitutively expressed and cotranscribed. Disruption of ccdA by transposon Tn5-mob caused inability to form functional c-type cytochromes. Convincing evidence for the requirement of ccdA in cytochrome c biogenesis was obtained from (i) physiological studies, (ii) complementation analysis, (iii) biochemical and immunochemical analysis, and (iv) sequence analysis of the deduced ccdA gene product.

Strain TP43 not only was impaired in aerobic thiosulfate oxidation or dissimilatory nitrite reduction but also exhibited significantly reduced cellular yields compared to the wild type. The reduction in cellular yields was evidence of a general defect in energy metabolism. This evidence was supported by the inability of strain TP43 to oxidize TMPD and the absence of any effect of antimycin A, an inhibitor of electron transfer to the bc₁ complex, which caused reduction in the cellular yield of the wild type but not in that of strain TP43. Without antimycin A the yield of strain TP43 was identical to that of the wild type grown in the presence of antimycin A. This was evidence that electron transfer was abolished from ubiquinone to the bc₁ complex and further to cytochrome c₅₅₁ (40).

Analysis of cytochromes revealed the complete absence of holocytochromes c, whereas apoproteins of c-type cytochromes were still formed in strain TP43, as demonstrated by the immunochemical detection of SoxD, the periplasmic c-type cytochrome which is part of the sulfur-oxidizing enzyme system in P. pantotrophus GB17 (37, 56). Moreover, the SoxD apoprotein was transported to the periplasm as demonstrated by its specific extraction and by immunochemical analysis. Therefore, disruption of ccdA eliminated an essential late step in cytochrome c biogenesis. This in turn affected energy conservation and caused reduced cellular yields.

To exclude a polar effect of the Tn5-mob which could have been responsible for the pleiotropy of strain TP43, the ccdA gene region was cloned into pVK101. TP43 harboring pVK1 was fully restored in its ability to form c-type cytochromes. This demonstrated that the inactivation of only ccdA caused the inability to form holo- c-type cytochromes, and thiol compounds could not compensate this defect as described for E. coli (42).

CcdA of P. pantotrophus is homologous to proteins of other bacteria involved in copper tolerance (17, 22), mercury resistance (47), spore synthesis (46), and cytochrome c biogenesis (13, 35, 45). CcdA was not cotranscribed with a thioredoxin in P. pantotrophus, and it missed the thioredoxin stretch at the C terminus as described for DipZ homologous proteins found in E. coli (13) or P. aeruginosa (35). CcdA homologues have been proposed to participate in class II cytochrome c biogenesis systems, whereas the class I cytochrome c biogenesis systems present in most gram-negative bacteria are proposed to involve a DipZ homologue (28). The participation of a CcdA homologue in the biogenesis of a class I cytochrome c has been described only for R. capsulatus (14) and CcdA of P. pantotrophus, closely related to R. capsulatus (31).

In analogy to previous findings we suggest that CcdA of P. pantotrophus transports electrons from the cytoplasm to the periplasm via the two cysteine residues, although its electron donor is still unknown. Such involvement has been suggested for CcdA of R. capsulatus (14) and for DsbD of E. coli (12, 21, 51).

The disruption of ccmG of P. denitrificans caused a partial deficiency in holo- c-type cytochromes, and it was proposed that CcmG participates in cytochrome c biogenesis by reducing disulfide bonds in the periplasm (34). However, it is still unknown in which way CcmG derives the reducing equivalents. On the basis of the data presented we suggest that CcdA may act in reducing CcmG directly or via an unknown mediator in P. pantotrophus GB17 which seems to play an important role in the rereduction of cytochrome c apoproteins in the periplasm prior to heme binding.

The primary structure of CcdA is 42% identical and 80% similar to that of the predicted ORF2 gene product of P. pantotrophus located within the sox gene cluster (20). ORF2, however, cannot complement the disrupted ccdA, as shown with strain TP43. Therefore, the possible ORF2 function is proposed to be different from that of ccdA; whether it is specific for or linked to lithotrophic sulfur oxidation remains to be analyzed.