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Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides.

ABSTRACT: Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain. cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically. We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension. Complementation of a cycA null mutant shows that there are at least two cycA promoters: one within 89 bp upstream of the translation initiation codon for a transcript beginning at -28, and at least one within 484 bp upstream for the remaining nine 5' ends. The 5' ends at -28 and -137 are more abundant in aerobically grown cells, while those at -38, -155, -250, and -300 are more abundant photosynthetically. DNA sequences with homology to the Escherichia coli sigma 70 consensus promoter sequence precede the 5' ends at -28 and -274, and there is weak homology upstream of the -82 and -250 ends.

SUBMITTER: MacGregor BJ

PROVIDER: S-EPMC208040 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides.

MacGregor B J BJ Donohue T J TJ

Journal of bacteriology 19910701 13

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain. cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically. We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension. Complementation of a cycA null mutant shows that there are at least two cy ...[more]

PMID: 1648072

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ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene.

Project description:Transcription of the Rhodobacter sphaeroides cytochrome c2 gene (cycA) is negatively regulated by both the presence of oxygen and intermediates in tetrapyrrole biosynthesis. A mutation responsible for uncoupling cycA transcription from tetrapyrrole availability was localized to a gene (chrR) that encodes a 357-amino-acid protein. Analysis of a defined chrR null mutation indicated that this protein positively regulated cycA transcription. From this and other results, it appeared that the positive action of ChrR on cycA transcription is blocked by altering the availability of either heme or some intermediate in tetrapyrrole biosynthesis. A single missense mutation which substitutes an Arg for a Cys at residue 182 of ChrR (C182R) was shown to be necessary and sufficient for the increased cycA transcription seen in the mutant strain Chr4. Thus, it appears that this C182R substitution generated an altered-function form of ChrR. In addition, by analyzing cycA transcription in delta ChrR strains, we showed that ChrR was not required for increased cycA transcription under anaerobic conditions. Instead, our results indicated that ChrR and the response regulator PrrA (J. M. Eraso and S. Kaplan, J. Bacteriol. 176:32-43, 1994) functioned independently at the upstream cycA promoter that is activated under anaerobic conditions.

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Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction.

Project description:The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.

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Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

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Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: role of interprotein hydrogen bonds in binding and electron transfer.

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Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure.

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| S-EPMC196149 | biostudies-other

Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase.

Project description:To characterize protein structures that control proton uptake, we assayed forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The N139D/D132N mutant contains a carboxyl group 6 Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow, but once subunit III is removed, its activity is the same as that of wild-type CcO lacking subunit III (∼1800 H+/s). Thus, a carboxyl group∼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cys-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO because of simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.

| S-EPMC3082432 | biostudies-literature

Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.

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| S-EPMC3342662 | biostudies-literature

Features of Rhodobacter sphaeroides CcmFH.

Project description:In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.

| S-EPMC145331 | biostudies-literature

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