Characterization of a new type of sulfite dehydrogenase from Paracoccus pantotrophus GB17

The periplasmic sulfite dehydrogenase of Paracoccus pantotrophus GB17 was purified to homogeneity by a four-step procedure from cells grown lithoautotrophically with thiosulfate. The molecular mass of native sulfite dehydrogenase was 190 kDa as determined by native gradient PAGE. SDS-PAGE showed sulfite dehydrogenase to comprise two subunits with molecular masses of 47 kDa and 50 kDa, suggesting an alpha2beta2 structure. The N-terminal amino acid sequence and immunochemical analysis using SoxC-specific antibodies identified the 47-kDa protein as the soxC gene product. SoxD-specific antibodies identified the 50-kDa protein as SoxD. Based on the molecular masses deduced from the nucleotide sequence for mature SoxC (43,442 Da) and SoxD (37,637 Da) sulfite dehydrogenase contained 1.30 mol molybdenum/mol alpha2beta2 sulfite dehydrogenase. The iron content was 3.17 mol/mol alpha2beta2 sulfite dehydrogenase, and 3.53 mol heme/mol alpha2beta2 sulfite dehydrogenase was determined by pyridine hemochrome analysis. These data are consistent with the two heme-binding domains (CxxCH), characteristic for c-type cytochromes, deduced from the soxD nucleotide sequence. Electrospray ionization revealed two masses for SoxC of 43,503 and 43,897 Da. The difference in molecular mass was attributed to the molybdenum cofactor of SoxC. For SoxD a mass of 38,815 Da was determined; this accounted for the polypeptide and two covalently bound hemes. Reconstitution of the catalytic activity of sulfite dehydrogenase required additional fractions; these eluted from Q Sepharose at 0.05, 0.25, and 0.30 M NaCl. The K(m) of sulfite dehydrogenase for sulfite was 7.0 microM and for cytochrome c 19 microM. Sulfite dehydrogenase activity was inhibited by sulfate and phosphate. The structural and catalytic properties make sulfite dehydrogenase from P. denitrificans GB17 distinct from sulfite oxidases of other prokaryotic or eukaryotic sources.     

Novel Genes of the sox Gene Cluster, Mutagenesis of the Flavoprotein SoxF, and Evidence for a General Sulfur-Oxidizing System in Paracoccus pantotrophus GB17

The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol. soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer. soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.     

A soxA gene, encoding a diheme cytochrome c, and a sox locus, essential for sulfur oxidation in a new sulfur lithotrophic bacterium

A mobilizable suicide vector, pSUP5011, was used to introduce Tn5-mob in a new facultative sulfur lithotrophic bacterium, KCT001, to generate mutants defective in sulfur oxidation (Sox(-)). The Sox(-) mutants were unable to oxidize thiosulfate while grown mixotrophically in the presence of thiosulfate and succinate. The mutants were also impaired in oxidizing other reduced sulfur compounds and elemental sulfur as evident from the study of substrate oxidation by the whole cells. Sulfite oxidase activity was significantly diminished in the cell extracts of all the mutants. A soxA gene was identified from the transposon-adjacent genomic DNA of a Sox(-) mutant strain. The sequence analysis revealed that the soxA open reading frame (ORF) is preceded by a potential ribosome binding site and promoter region with -10- and -35-like sequences. The deduced nucleotide sequence of the soxA gene was predicted to code for a protein of 286 amino acids. It had a signal peptide of 26 N-terminal amino acids. The amino acid sequence showed similarity with a putative gene product of Aquifex aeolicus, soluble cytochrome c(551) of Chlorobium limicola, and the available partial SoxA sequence of Paracoccus denitrificans. The soxA-encoded product seems to be a diheme cytochrome c for KCT001 and A. aeolicus, but the amino acid sequence of C. limicola cytochrome c(551) revealed a single heme-binding region. Another transposon insertion mutation was mapped within the soxA ORF. Four other independent transposon insertion mutations were mapped in the 4.4-kb soxA contiguous genomic DNA region. The results thus suggest that a sox locus of KCT001, essential for sulfur oxidation, was affected by all these six independent insertion mutations.     

Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase

The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T. P., & Hopper, D. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4626-4630], however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit. In this paper we report data confirming the conclusions of X-ray crystallographic analysis. From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250. These values have been confirmed by gel electrophoresis under denaturing conditions. Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer. The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes. The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported. The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.     

Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans

Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.     

Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17.

The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19). The corresponding wild-type region was cloned previously (G. Mittenhuber, K. Sonomoto, M. Egert, and C. G. Friedrich, J. Bacteriol. 173:7340-7344, 1991). Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB. This polypeptide had an M(r) of 60.573, including a possible signal peptide. The function of the SoxB protein of P. denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus. The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P. denitrificans GB17.     

Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria.

Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.     

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.     

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment.

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.     

Purification of Thiobacillus denitrificans siroheme sulfite reductase and investigation of some molecular and catalytic properties.

A siroheme-containing sulfite reductase was isolated from Thiobacillus denitrificans, purified to an electrophoretically homogenous state, and investigated with regard to some of its molecular and catalytic properties. The enzyme was a tetramer with a molecular weight of 160 000, consisting of two types of subunits arranged to an alpha 2 beta 2-structure. The molecular weight of the alpha-subunit was 38 000, that of the beta-subunit 43 000. As prosthetic groups siroheme and Fe/S groupings could be detected. The absorption spectrum showed maxima at 273 nm, 393 nm, and 594 nm; the molar extinction coefficient at these wavelengths were 280, 181, and 60 . 10(3) cm2 . mmol-1, respectively. With reduced viologen dyes the enzyme reduced sulfite to sulfide, thiosulfate and trithionate. In many properties T. denitrificans sulfite reductase closely resembled desulfoviridin, the dissimilatory sulfite reductase of Dssulfovibrio species. It is proposed that the physiological function of this enzyme is not to reduce but rather to form sulfite from reduced sulfur compounds in the course of dissimilatory sulfur oxidation in T. denitrificans.     

Sulfite:Cytochrome c oxidoreductase from Thiobacillus novellus. Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC ). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alphabeta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c(552) subunit (midpoint redox potential, E(m8.0) = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K(m) values for sulfite and cytochrome c(550) were determined to be 27 and 4 micrometer, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.     

Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.     

Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

MauG,a novel diheme protein required for tryptophan tryptophylquinone biogenesis

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH. A system was developed for the homologous expression of MauG in P. denitrificans. Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction. The protein contains two covalent c-type hemes, as predicted from the deduced sequence. EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry. The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity. The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases. MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor. Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen. The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis.     

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.     

Expression in periplasmic space of Shewanella oneidensis

A Shewanella expression system has been used for an overproduction of c-type multiheme proteins. The proteins were exported to the periplasmic space for the maturation. Since the periplasmic expression system is attractive, especially for protease-sensitive proteins, an expression vector containing a signal peptide was constructed for expressions in the periplasmic space of Shewanella oneidensis. To evaluate the system, two eukaryotic proteins which originally do not have signal sequences and are difficult to express in Escherichia coli, were selected. The first is human cytochrome c. Properties of the recombinant cytochrome c were identical to those previously reported, indicating the protein is intact. The other was potato calcium-dependent protein kinase. The protein was expressed in periplasmic space. These results indicated that the system is generally applicable for any protein expression including c-type cytochromes, protease-sensitive proteins and those with multi-disulfide bonds because of transportation to the periplasmic space.     

Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects.

By using synthetic oligonucleotides, the gene encoding soluble cytochrome c550 was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the mature protein was found to be similar to the primary structure of purified cytochrome c550 except for the presence of seven additional amino acid residues at the C terminus. At the N terminus of the primary structure was found an additional stretch of 19 amino acid residues that had the typical features of the signal sequence of the cytochrome. Comparison of the nucleotide sequences of the upstream regions of the P. denitrificans cytochrome c550 gene and bc1 operon revealed three regions with a distinct organization that showed strong similarity. Downstream of the c550 gene was found part of another gene, the deduced amino acid sequence of which showed strong homology with subunit 1 of the cytochrome aa3 oxidase. For gene replacement experiments, the suicide vector pGRPd1 was constructed. The cytochrome c550 gene was inactivated by insertion of a kanamycin resistance gene, and the mutated gene was cloned into this vector. Recombination with the wild-type gene resulted in a mutant strain with an inactivated cytochrome gene. Isolated mutant strains were unable to synthesize the soluble cytochrome, as judged by spectrum analysis and analysis of periplasmic proteins by gel electrophoresis and heme staining. The mutation resulted in a 14% decrease in the growth yield during aerobic heterotrophic growth and in a 40% decrease in the maximum specific growth rate during growth on methylamine. Furthermore, a longer lag phase was observed under both growth conditions. The mutation had no effect on growth yield, maximum specific growth rate, and duration of the lag phase during anaerobic growth in the presence of nitrate. In addition, there was no accumulation of nitrite and nitrous oxide.