The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases

Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria.     

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.     

sacB-5-Fluoroorotic acid-pyrE-based bidirectional selection for integration of unmarked alleles into the chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB-5-fluoroorotic acid (5FOA)--pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura-) and hence resistant to 5FOA (5FOA(r)). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA(r) to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA(r) selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA(r) colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

Importance of a conserved hydrogen-bonding network in cytochromes c to their redox potentials and stabilities.

To understand the determinants of redox potential and protein stability in c-type cytochromes, we have characterized two mutations to a highly conserved tyrosine group, tyrosine-75, of Rhodobacter capsulatus cytochrome c2. Mutant Y75F was designed to test the importance of the tyrosine hydroxyl group to the typically high redox potentials of the cytochromes c2 while maintaining a hydrophobic core. Mutant Y75C was designed to test the importance of a large hydrophobic group to redox potential by replacing an aromatic group with a small nonpolar group. Both mutants exhibit spectral and redox properties indicating that their heme environments have been perturbed. The kinetics of reduction by lumiflavin semiquinone and photooxidation by Rhodobacter sphaeroides photosynthetic reaction centers have been used to demonstrate that both mutants are structurally analogous to the wild-type protein at the active site of electron transfer. Different degrees of relative stability of the mutants toward a denaturant have been observed with the order being Y75C less than wt less than Y75F in the oxidized state and Y75C less than Y75F less than wt in the reduced state. These results are discussed in light of the recent structure determination of the R. capsulatus wild-type ferrocytochrome c2 to suggest that R. capsulatus tyrosine-75, or its equivalent in other species, is part of a conserved hydrogen-bonding network which plays an important role in maintaining high redox potentials and protein stability of cytochromes c in general.     

Ornithine lipid is required for optimal steady-state amounts of c-type cytochromes in Rhodobacter capsulatus

The c-type cytochromes are haemoproteins that are subunits or physiological partners of electron transport chain components, like the cytochrome bc(1) complex or the cbb(3)-type cytochrome c oxidase. Their haem moieties are covalently attached to the corresponding apocytochromes via a complex post-translational maturation process. During our studies of cytochrome biogenesis, we uncovered a novel class of mutants that are unable to produce ornithine lipid and that lack several c-type cytochromes. Molecular analyses of these mutants led us to the ornithine lipid biosynthesis genes of Rhodobacter capsulatus. Herein, we have characterized these mutants, and established the chemical structure of this non-phosphorus membrane lipid from R. capsulatus. Ornithine lipids are known to induce potent host immune responses, including B-lymphocyte mitogenicity, adjuvanticity and macrophage activation. Yet, despite their widespread occurrence in Eubacteria, and the diverse biological effects they elicit in mammals, their physiological role in bacterial cells remained hitherto poorly defined. Our findings now indicate that under certain bacterial growth conditions ornithine lipids are crucial for optimal steady-state amounts of some extracytoplasmic proteins, including several c-type cytochromes, and attribute them a novel and important biological function.     

DNA gyrase activities from Rhodobacter capsulatus: analysis of target(s) of coumarins and cloning of the gyrB locus

Bacterial DNA gyrase is composed of two subunits, gyrase A and B, and is responsible for negatively supercoiling DNA in an ATP-dependent manner. The coumarin antibiotics novobiocin and coumermycin are known inhibitors of bacterial DNA gyrase in vivo and in vitro. We have cloned, mapped, and partially sequenced Rhodobacter capsulatus gyrB which encodes the gyrase B subunit that is presumably involved in binding to coumarins. DNA gyrase activities from crude extracts of R. capsulatus were detected and it was shown that the R. capsulatus activity is (1) inhibited by novobiocin and coumermycin, (2) ATP-dependent and, (3) present in highly aerated and anaerobically grown cells. We previously observed that when R. capsulatus coumermycin-resistant strains are continuously recultured on media containing coumermycin they sometimes acquired mutations in hel genes (i.e., cytochromes c biogenesis mutations). We discuss the possibility that coumarins may inhibit cytochromes c biogenesis as a second target in R. capsulatus via hel (i.e., a putative ATP-dependent heme exporter).     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.     

Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes

Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.     

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.     

The dithiol:disulfide oxidoreductases DsbA and DsbB of Rhodobacter capsulatus are not directly involved in cytochrome c biogenesis,but their inactivation restores the cytochrome c biogenesis defect of CcdA-null mutants

The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli, are thought to supply the reducing equivalents required for the biogenesis of c-type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA-null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps(-)) and do not produce any active cytochrome c oxidase (Nadi(-)) due to a pleiotropic cytochrome c deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps(+) Nadi(+) colonies that produce c-type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps(+) growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a dsbA ccdA double mutant was shown to be Ps(+) Nadi(+), establishing that in R. capsulatus the inactivation of dsbA suppresses the c-type cytochrome deficiency due to the absence of ccdA. Next, the ability of the wild-type dsbA allele to suppress the Ps(+) growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA-independent ccdA revertants. Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps(+) phenotypes could be suppressed by the wild-type allele of dsbB. As with dsbA, a dsbB ccdA double mutant was also Ps(+) Nadi(+) and produced c-type cytochromes. Therefore, the absence of either DsbA or DsbB restores c-type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of R. capsulatus are Ps(+) and produce c-type cytochromes, unlike their E. coli counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in R. capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions.     

Identification of a gene encoding a thioredoxin-like product necessary for cytochrome c biosynthesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

A Tn5-induced mutant of Rhizobium leguminosarum bv. viciae could not form nitrogen-fixing nodules on pea or vetch because of a lesion in electron transport to oxygen. The mutant lacked spectroscopically detectable cytochromes c and aa3. No proteins containing c-type cytochrome could be identified in the mutant by heme staining of proteins fractionated on polyacrylamide gels, indicating that the mutant was defective in maturation of all c-type cytochromes. The Tn5 mutation was determined to be located in a gene that was called cycY. The cycY gene product is homologous to the thioredoxin-like protein HelX involved in the assembly of c-type cytochromes in Rhodobacter capsulatus and to an open reading frame from a Bradyrhizobium japonicum gene cluster containing other genes involved in cytochrome c biogenesis. Our observations are consistent with CycY functioning as a thioredoxin that reduces cysteine residues in apocytochromes c before heme attachment.     

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.     

Membrane-spanning and periplasmic segments of CcmI have distinct functions during cytochrome c Biogenesis in Rhodobacter capsulatus

In gram-negative bacteria, like Rhodobacter capsulatus, about 10 membrane-bound components (CcmABCDEFGHI and CcdA) are required for periplasmic maturation of c-type cytochromes. These components perform the chaperoning and thio-oxidoreduction of the apoproteins as well as the delivery and ligation of the heme cofactors. In the absence of any of these components, including CcmI, proposed to act as an apocytochrome c chaperone, R. capsulatus does not have the ability to produce holocytochromes c or consequently to exhibit photosynthetic growth and cytochrome cbb3 oxidase activity. Previously, we have demonstrated that null mutants of CcmI partially overcome cytochrome c deficiency phenotypes upon overproduction of the CcmF-R. capsulatus CcmH (CcmF-CcmH(Rc)) couple in a growth medium-dependent manner and fully bypass these defects by additional overproduction of CcmG. Here, we show that overproduction of the CcmF-CcmH(Rc) couple and overproduction of the N-terminal membrane-spanning segment of CcmI (CcmI-1) have similar suppression effects of cytochrome c maturation defects in CcmI-null mutants. Likewise, additional overproduction of CcmG, the C-terminal periplasmic segment of CcmI (CcmI-2), or even of apocytochrome c2 also provides complementation abilities similar to those of these mutants. These results indicate that the two segments of CcmI have different functions and support our earlier findings that two independent steps are required for full recovery of the loss of CcmI function. We therefore propose that CcmI-1 is part of the CcmF-CcmH(Rc)-dependent heme ligation, while CcmI-2 is involved in the CcdA- and CcmG-dependent apoprotein thioreduction steps, which intersect at the level of CcmI during cytochrome c biogenesis.     

Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c

Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.     

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.     

petR, located upstream of the fbcFBC operon encoding the cytochrome bc1 complex, is homologous to bacterial response regulators and necessary for photosynthetic and respiratory growth of Rhodobacter capsulatus

Interposon mutagenesis of a region upstream of the petABC(fbcFBC) operon, encoding the ubiquinol: cytochrome c2 oxidoreductase (bc1 complex) of the photosynthetic bacterium Rhodobacter capsulatus revealed the presence of two genes, petP and petR. DNA nucleotide sequence determination of this region indicated that petP and petR are transcribed in the same direction as the petABC(fbcFBC) operon, and are translationally coupled. A silent insertion located in the interoperonal region separating petPR and the petABC(fbcFBC) genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putative petR gene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth of R. capsulatus.     

Positive and negative selection of mutant forms of prokaryotic (cyanobacterial) ribulose-1,5-bisphosphate carboxylase/oxygenase

A system for biological selection of randomly mutagenized ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) genes from the cyanobacterium Synechococcus PCC6301 was designed in which a Rubisco deletion mutant of the photosynthetic bacterium Rhodobacter capsulatus served as a host. Trans-complementation with the Synechococcus PCC6301 rbcLS genes enabled anaerobic photoautotrophic growth of the R.capsulatus deletion strain with 5% CO(2), but not with 1.5% CO(2) in the atmosphere, and this strain could not grow under aerobic chemoautotrophic conditions. Phenotypic differences between the R.capsulatus host strain complemented with the wild-type rbcLS genes and transconjugates carrying mutated genes were used to identify mutants that were able to complement to photoautotrophic growth with 1.5% CO(2). These "positive" mutant proteins were unaffected for any measured kinetic properties, with a single exception. A mutant with a valine substitution at phenylalanine 342 had an increased affinity for ribulose-1,5-bisphosphate. Mutants with changes in the affinity for CO(2) were isolated through negative selection, in which mutants incapable of complementing R.capsulatus to photoautotrophic growth with 5% CO(2) were identified. Mutations at aspartate 103 resulted in enzymes that were greatly affected for different kinetic parameters, including an increased K(m) for CO(2). This study demonstrated that random mutagenesis and bioselection procedures could be used to identify mutations that influence important properties of bacterial Rubisco; these residues would not have been identified by other methods.     

Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus

The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c1 and b of the cytochrome bc1 complex, alone or in combination with deletion of the gene for cytochrome c2. Deletion of cytochrome c1 renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c2, because in the absence of the bc1 complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c2 is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc1 complex. The deletion of cytochromes c1 and c2 reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with Em7 = +312 mV and Em6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.     

Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration.

We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F.E. Jenney and F. Daldal, EMBO J. 12:1283-1292, 1993; F.E. Jenney, R.C. Prince, and F. Daldal, Biochemistry 33:2496-2502, 1994). One pathway is via the well-characterized, water-soluble cytochrome c2 (cyt c2), and the other is via a novel membrane-associated c-type cytochrome named cyt cy. In this work, we probed the role of cyt cy in respiratory electron transport by isolating a set of R. capsulatus mutants lacking either cyt c2 or cyt cy, in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway. The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt cy, like cyt c2, connects the bc1 complex and the cyt c oxidase during respiratory electron transport. Whether cyt c2 and cyt cy are the only electron carriers between these two energy-transducing membrane complexes of R. capsulatus is unknown.