Cloning and overexpression of the Rhodobacter capsulatus hemH gene.

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34?kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R.?capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34?kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R.?capsulatus cultures.     

Nucleotide sequence of the Rhodobacter capsulatus hemH gene

The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence     

Isolation of Rhodobacter capsulatus transketolase: Cloning and sequencing of its structural tktA gene

Rhodobacter capsulatus transketolase (Tkt) protein has been isolated from strain B10 by heparin affinity chromatography. Oligodeoxyribonucleotides (oligo) constructed as based on the amino-acid sequences were used for polymerase chain reaction (PCR) amplification on total genomic DNA. Southern hybridization with the PCR product as a probe allowed the isolation of a 5-kb PstI DNA fragment containing the structural Tkt-encoding gene (tktA) which was cloned and sequenced. The deduced tktA product of 671 aa (72 815 Da) shares 59% identity with Rhodobacter sphaeroides Tkt     

Cloning and characterization of the ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.     

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.     

Nucleotide transport in Rhodobacter capsulatus.

Cytoplasmic membrane vesicles isolated from the gram-negative photosynthetic bacterium Rhodobacter capsulatus catalyzed the transport of nucleotides. No transport occurred in the intact bacteria unless they were pretreated with EDTA. The transport rate was measured by incorporation of radioactive phosphate into externally added ADP or by incorporation of nonradioactive phosphate into added labeled ADP. The catalytic activities which utilized the added ADP were photosynthetic ATP synthesis, Pi-ADP exchange, and adenylate kinase. These activities were shown to occur on the cytoplasmic side of the internal membrane. The products were found in the outer medium. The rate of nucleotide transport across the membranes was comparable to the rate of photophosphorylation. These results indicated that nucleotides can be transported across the cytoplasmic membrane but not across the outer membrane of the native R. capsulatus cell. Therefore, by analogy to the mitochondrial ATP-ADP translocator, the exchange might function as an energy transfer system to the periplasm of these bacteria.     

Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus.

The purple nonsulfur photosynthetic eubacterium Rhodobacter capsulatus is a versatile organism that can obtain cellular energy by several means, including the capture of light energy for photosynthesis as well as the use of light-independent respiration, in which molecular oxygen serves as a terminal electron acceptor. In this study, we have identified and characterized a novel gene, senC, mutations in which affect respiration as well as the induction of photosynthesis gene expression. The protein coded by senC exhibits 33% sequence identity to the yeast nucleus-encoded protein SCO1, which is thought to be a mitochondrion-associated cytochrome c oxidase assembly factor. Like yeast SCO1, SenC is required for optimal cytochrome c oxidase activity in aerobically grown R. capsulatus cells. We further show that senC is required for maximal induction from the puf and puh operons, which encode the structural polypeptides of the light-harvesting and reaction center complexes.