Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro. Correlation with photoinhibition studies.

1. Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm. The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen. The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived. Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex. 2. Cells of R. capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction. A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant. The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131). In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2] were ineffective as mediators of electrons from isoascorbate. Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide. 3. Purified ferroycytochrome c2 from R. capsulatus was oxidised by nitrous oxide in the presence of periplasm from R. capsulatus MTG4/S4. The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates. The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl. 4. Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase. However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase. The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction. 5. Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.     

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.     

Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae

Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.     

Rhodobacter capsulatus CycH: a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes.

While searching for components of the soluble electron carrier (cytochrome c2)-independent photosynthetic (Ps) growth pathway in Rhodobacter capsulatus, a Ps- mutant (FJM13) was isolated from a Ps+ cytochrome c2-strain. This mutant could be complemented to Ps+ growth by cycA encoding the soluble cytochrome c2 but was unable to produce several c-type cytochromes. Only cytochrome c1 of the cytochrome bc1 complex was present in FJM13 cells grown on enriched medium, while cells grown on minimal medium contained at various levels all c-type cytochromes, including the membrane-bound electron carrier cytochrome cy. Complementation of FJM13 by a chromosomal library lacking cycA yielded a DNA fragment which also complemented a previously described Ps- mutant, MT113, known to lack all c-type cytochromes. Deletion and DNA sequence analyses revealed an open reading frame homologous to cycH, involved in cytochrome c biogenesis. The cycH gene product (CycH) is predicted to be a bipartite protein with membrane-associated amino-terminal (CycH1) and periplasmic carboxyl-terminal (CycH2) subdomains. Mutations eliminating CyCH drastically decrease the production or all known c-type cytochromes. However, mutations truncating only its CycH2 subdomain always produce cytochrome c1 and affect the presence of other cytochromes to different degrees in a growth medium-dependent manner. Thus, the subdomain CycH1 is sufficient for the proper maturation of cytochrome c1 which is the only known c-type cytochrome anchored to the cytoplasmic membrane by its carboxyl terminus, while CycH2 is required for efficient biogenesis of other c-type cytochromes. These findings demonstrate that the two subdomains of CycH play different roles in the biogenesis of topologically distinct c-type cytochromes and reconcile the apparently conflicting data previously obtained for other species.     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation.

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence of the flavoprotein of flavocytochrome c of Chromatium vinosum, suggesting the involvement of the flavoprotein in thiosulfate oxidation of P. denitrificans GB17.     

Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes.

We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration.

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.     

The 4784V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermoiability in initiation of Escherichia coli DNA replication

The prototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb Pstl fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcali-genes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90kDa catalytic subunit (NAPA) and a 15kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.     

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.     

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.     

HCF164 Encodes a Thioredoxin-Like Protein Involved in the Biogenesis of the Cytochrome b6f Complex in Arabidopsis

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.     

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Ω) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases. Received: 12 August 1996 / Accepted: 29 October 1996     

The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).