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Organization and expression of the Rhodobacter sphaeroides cycFG operon.

ABSTRACT: The Rhodobacter sphaeroides cycFG operon has been cloned, sequenced, and mapped to approximately coordinate 2500 of chromosome I. The cycF gene encodes cytochrome c554, a member of the class II family of soluble cytochrome c proteins. The cycF open reading frame includes a 20-amino acid extension at its N terminus which has not been detected in cytochrome c554. Antiserum against cytochrome c554 shows that this protein is localized to the periplasm of wild-type cells, which suggests that this N-terminal extension functions as a signal peptide. The predicted cycG gene product is a diheme cytochrome c with a subunit molecular mass of approximately 32 kDa. While a cytochrome with the properties predicted for CycG has not been reported for R. sphaeroides, we have tentatively identified this protein as a heme-staining polypeptide that is associated with membranes. CycG could have an overall structure similar to that of several other electron carriers, since the similarity between the predicted amino acid sequence of CycG and other multiheme cytochrome c proteins extends throughout the polypeptide. The cycFG transcript is approximately 1,500 nucleotides long and has a single 5' end 26 nucleotides upstream of the start of cycF translation. Expression of cycFG is regulated at the level of mRNA accumulation, since approximately fivefold-higher levels of both cycF-specific transcript and cytochrome c554 protein are detected in cell extracts from aerobic cultures in comparison with those from anaerobically grown cells. Although cytochrome c554 was detected under all growth conditions tested, the highest levels of this protein were found when cells generate energy via aerobic respiration.

SUBMITTER: Flory JE

PROVIDER: S-EPMC177178 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Organization and expression of the Rhodobacter sphaeroides cycFG operon.

Flory J E JE Donohue T J TJ

Journal of bacteriology 19950801 15

The Rhodobacter sphaeroides cycFG operon has been cloned, sequenced, and mapped to approximately coordinate 2500 of chromosome I. The cycF gene encodes cytochrome c554, a member of the class II family of soluble cytochrome c proteins. The cycF open reading frame includes a 20-amino acid extension at its N terminus which has not been detected in cytochrome c554. Antiserum against cytochrome c554 shows that this protein is localized to the periplasm of wild-type cells, which suggests that this N-t ...[more]

PMID: 7543472

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Project description:BackgroundThe genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data.ResultsThe analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles.ConclusionsOur analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. We identified R. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in R. sphaeroides whereas modules related to RcGTA production and signalling showed lack of preservation in R. sphaeroides. In addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mRNA expression and protein abundance data for which simple correlation measurements have previously had mixed results.

Project description:The Q gene of the facultative photoheterotroph Rhodobacter sphaeroides, localized immediately upstream of the oxygen- and light-regulated puf operon, encodes a 77-amino-acid polypeptide. The 5' and 3' ends of the 561-bp Q transcript were determined. To gain insight into the role of the Q gene product, a number of Q mutations were constructed by oligonucleotide-directed mutagenesis and subsequent substitution of the mutated form of the gene in single copy for the chromosomal copy via homologous recombination. The resulting mutants can grow photosynthetically, with the exception of QSTART, in which the initiation codon for the Q protein was altered. Spectral analysis of the intracytoplasmic membranes showed that one of the missense mutants (QdA) was deficient in the formation of detectable B875 light-harvesting complex (LHC), whereas deletion of the stem-loop structure (Qloop) failed to form B800-850 LHC when grown anaerobically either in the dark or under light intensity of 100 W/m2. Other missense mutants (QuA and QuB) contained either more B800-850 LHC or more B875 LHC, respectively, than the wild type. Although the levels of puf and puc transcripts isolated from QSTART grown anaerobically on succinate-dimethyl sulfoxide in the dark were comparable to wild-type levels, no B875 spectral complex was detected and there was a greater than 90% reduction in the level of the B800-850 pigment-protein complex. It has also been confirmed that the ultimate cellular levels of either the B875 or B800-850 spectral complexes can vary over wide limits without any change in the level(s) of complex specific transcripts. When the wild-type Q gene was reintroduced in trans into the Q mutations, QSTART was able to grow photosynthetically and both B800-850 and B875 spectral complexes were formed in either QdA or Qloop. Finally, we demonstrated that the level of each puf-specific mRNA behaves independently of one another as well as independently of the level(s) of Q gene-specific mRNA. These results are compatible with the existence of regulatory sequences affecting the puf mRNA level(s) being localized within the Q structural gene. These results suggest that Q-specific expression is uncoupled from puf-specific transcription and that the Q protein is not involved in the regulation of transcription of the puf operon but is directly involved in the assembly of both the B875 and B800-850 pigment-protein complexes.

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Organization and expression of the Rhodobacter sphaeroides cycFG operon.

Publications

Organization and expression of the Rhodobacter sphaeroides cycFG operon.

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