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 role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.     

Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

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.     

Isolation of mutants and genes involved in cytochromes c biosynthesis in Rhodobacter capsulatus.

Mutants of the photosynthetic bacterium Rhodobacter capsulatus that have combined deficiencies in the cytochrome b/c1 complex and other c-type cytochromes have been isolated. These mutants were unable to grow anaerobically in the light or dark but could grow aerobically. Cosmids with R. capsulatus wild-type DNA that complement the mutants have been used to construct genetic and physical maps of the affected genes. Complementation profiles with Tn5 and mini-Mu insertions in these cosmids and subcloned fragments from them indicated that at least three genes (called helA, helB, and helC) are involved in the defects in cytochromes c biosynthesis. The genes are clustered, and helC is transcribed away from helA and helB. Stable insertion mutants in each gene were constructed. It is postulated that helA, helB, and helC are involved in posttranslational processing during cytochromes c synthesis.     

Overproduction of CcmG and CcmFH(Rc) fully suppresses the c-type cytochrome biogenesis defect of Rhodobacter capsulatus CcmI-null mutants

Gram-negative bacteria like Rhodobacter capsulatus use intertwined pathways to carry out the posttranslational maturation of c-type cytochromes (Cyts). This periplasmic process requires at least 10 essential components for apo-Cyt c chaperoning, thio-oxidoreduction, and the delivery of heme and its covalent ligation. One of these components, CcmI (also called CycH), is thought to act as an apo-Cyt c chaperone. In R. capsulatus, CcmI-null mutants are unable to produce c-type Cyts and thus sustain photosynthetic (Ps) growth. Previously, we have shown that overproduction of the putative heme ligation components CcmF and CcmH(Rc) (also called Ccl1 and Ccl2) can partially bypass the function of CcmI on minimal, but not on enriched, media. Here, we demonstrate that either additional overproduction of CcmG (also called HelX) or hyperproduction of CcmF-CcmH(Rc) is needed to completely overcome the role of CcmI during the biogenesis of c-type Cyts on both minimal and enriched media. These findings indicate that, in the absence of CcmI, interactions between the heme ligation and thioreduction pathways become restricted for sufficient Cyt c production. We therefore suggest that CcmI, along with its apo-Cyt chaperoning function, is also critical for the efficacy of holo-Cyt c formation, possibly via its close interactions with other components performing the final heme ligation steps during Cyt c biogenesis.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Oxidation-reduction properties of disulfide-containing proteins of the Rhodobacter capsulatus cytochrome c biogenesis system

Oxidation-reduction titrations for the active-site disulfide/dithiol couples of the helX- and ccl2-encoded proteins involved in cytochrome c biogenesis in the purple non-sulfur bacterium Rhodobacter capsulatus have been carried out. The R. capsulatus HelX and Ccl2 proteins are predicted to function as part of a dithiol/disulfide cascade that reduces a disulfide on the apocytochromes c so that two cysteine thiols are available to form thioether linkages between the heme prosthetic group and the protein. Oxidation-reduction midpoint potential (E(m)) values, at pH 7.0, of -300 +/- 10 and -210 +/- 10 mV were measured for the HelX and Ccl2 (a soluble, truncated form of Ccl2) R. capsulatus proteins, respectively. Titrations of the disulfide/dithiol couple of a peptide designed to serve as a model for R. capsulatus apocytochrome c(2) have also been carried out, and an E(m) value of -170 +/- 10 mV was measured for the model peptide at pH 7.0. E(m) versus pH plots for HelX, Ccl2, and the apocytochrome c(2) model peptide were all linear over the pH range from 5.0 to 8.0, with the -59 mV/pH unit slope expected for a reaction in which two protons are taken up for each disulfide that is reduced. These results provide thermodynamic support for the proposal that HelX reduces Ccl2 and that reduced Ccl2, in turn, serves as the reductant for the production of the two thiols of the CysXxxYyyCysHis heme-binding motif of the apocytochromes.     

Expression of regulatory nif genes in Rhodobacter capsulatus.

Translational fusions of the Escherichia coli lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nif gene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and nifR4 (the analog of ntrA in Klebsiella pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and nifR4 were not expressed under these conditions. The implications for the regulatory circuit of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph.     

A novel membrane-associated c-type cytochrome, cyt cy, can mediate the photosynthetic growth of Rhodobacter capsulatus and Rhodobacter sphaeroides.

Mutants of Rhodobacter capsulatus lacking the soluble electron carrier cytochrome c2 are able to grow photosynthetically (Ps+), whereas Rhodobacter sphaeroides is unable to do so. To understand this unusual electron transfer pathway the gene required for cyt c2-independent growth of R.capsulatus was sought using chromosomal libraries derived from a cyt c2- mutant of this species to complement a Ps- cyt c2- mutant of R.sphaeroides to Ps+ growth. The complementing 1.2 kbp DNA fragment contained a gene, cycY, encoding a novel membrane-associated c-type cytochrome, cyt cy, based on predicted amino acid sequence, optical difference spectra and SDS-PAGE analysis of chromatophore membranes. The predicted primary sequence of cyt cy is unusual in having two distinct domains, a hydrophobic amino-terminal region and a carboxyl-terminus with strong homology to cytochromes c. A cyt cy- mutant of R.capsulatus remains Ps+ as does the cyt c2- mutant. However, a mutant lacking both cyt c2 and cy is Ps-, and can be complemented to Ps+ by either cyt c2 or cyt cy. These findings demonstrate that each of the cytochromes c2 and cy is essential for photosynthesis only in the absence of the other. Thus, two distinct electron transfer pathways, unrecognized until now, operate during photosynthesis in R.capsulatus under appropriate conditions, one via the soluble cyt c2 and the other via the membrane-associated cyt cy.     

Clustering of genes necessary for hydrogen oxidation in Rhodobacter capsulatus.

Three cosmids previously shown to contain information necessary for the expression of uptake of hydrogenase in Rhodobacter capsulatus were found to be present in a cluster on the chromosome. Earlier genetic experiments suggested the presence of at least six genes essential for hydrogenase activity that are now shown to be in a region of approximately 18 kb that includes the structural genes for the enzyme. A potential response regulator gene was sequenced as a part of the hup gene region.     

Biochemical requirements for the maturation of mitochondrial c-type cytochromes

Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.     

Identification of a gene required for the oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus

The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild-type chromosome or by addition of 10μg I^-1 vitamin B₁₂ to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.     

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3′ end of cycH were termed cycJ, cycK and cycL. A deletion mutant (ΔcycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an M_r of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.     

Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins

As an extension of previous work from this laboratory using Clostridium pasteurianum flavodoxin [Tollin, G., Cheddar, G., Watkins, J. A., Meyer, T. E., & Cusanovich, M. A. (1984) Biochemistry 23, 6345-6349], we have measured the rate constants as a function of ionic strength for electron transfer from the semiquinones of Clostridium MP, Anacystis nidulans, and Azotobacter vinelandii flavodoxins to the following oxidants: cytochrome c from tuna and horse, Paracoccus denitrificans cytochrome c2, Pseudomonas aeruginosa cytochrome c-551, and ferricyanide. The rate constants extrapolated to infinite ionic strength (k infinity) for the C. MP flavodoxin are all slightly smaller than for the C. pasteurianum flavodoxin, as would be predicted on the basis of the higher redox potential of the C. MP protein. This indicates that there is a close similarity between the surface topographies of the two proteins in the vicinity of the coenzyme binding site. Moreover, the electrostatic interactions between the two flavodoxins and the various oxidants are also approximately the same. These studies justify our previous use of the crystallographic structure of the C. MP flavodoxin to interpret kinetic results obtained with the structurally uncharacterized C. pasteurianum flavodoxin. Despite their lower redox potentials, both Anacystis and Azotobacter flavodoxins are appreciably less reactive toward all of these oxidants (as much as 2 orders of magnitude in some cases) than are the Clostridium flavodoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced nitrogenase activity in strains of Rhodobacter capsulatus that overexpress the rnf genes

In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.     

Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway.

Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.