Organization and expression of the Rhodobacter sphaeroides cycFG operon

Flory, Janice E.; Donohue, Timothy J.

doi:10.1128/jb.177.15.4311-4320.1995

Cited by 9 publications

(10 citation statements)

References 53 publications

(48 reference statements)

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“…2), it appears that strains lacking only CcmH had ϳ30-fold less of these proteins (ϳ0.01 nmol/mg of soluble protein) than aerobically grown wild-type cells (ϳ0.36 nmol/mg). Consistent with this finding, Western blotting using antibodies to either cytochrome c 2 (3) or cytochrome c 554 (16) showed that each protein was at least 10-fold less abundant in cells lacking only CcmH compared to wild-type cells (data not shown).…”

Section: Vol 185 2003 R Sphaeroides Ccmfh 425supporting

confidence: 71%

Features of Rhodobacter sphaeroides CcmFH

2003

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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 ␤-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.

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Section: Vol 185 2003 R Sphaeroides Ccmfh 425supporting

confidence: 71%

Features of Rhodobacter sphaeroides CcmFH

2003

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“…We also tested whether a previously noted inverted repeat (centred at -24.5) regulated cycFG promoter function (Flory & Donohue, 1995). When a mutation that destroyed the downstream half-site (without changing the upstream half-site that overlaps a potential -35 element; Fig.…”

Section: An Inverted Repeat Overlapping the Cycfg Promoter Is Not Essmentioning

confidence: 99%

“…To test if such differential expression of aerobic cytochromes occurs in R. sphaeroides , we examined transcription of two cyt aa 3 genes ( ctaD and coxII) , both in steady-state cultures grown at different oxygen tensions and when photosynthetic (anaerobic) cultures were shifted to high (30 %) oxygen conditions. Since previous work suggested that the elevated levels of cytochrome c 554 found under high (30 %) oxygen growth conditions reflects increased cycFG transcription (Flory & Donohue, 1995), we also analysed function of this promoter. In considering how these genes might be regulated by changes in oxygen tension, the existence of sites similar to the consensus sequence for the Escherichia coli global anaerobic regulator FNR in the cycFG, coxII and ctaD control regions (Cao et al , 1991; Shapleigh & Gennis, 1992; Flory & Donohue, 1995) make it possible that their expression is repressed under anaerobic conditions by the R. sphaeroides FNR homologue, FnrL (Zeilstra-Ryalls & Kaplan, 1995).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides

Flory

Donohue

1997

Microbiology

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To decipher how the synthesis of energy-transducing enzymes responds to environmental cues, the response of three Rhodobacter sphaeroides aerobic cytochrome gene promoters was analysed under different conditions. Two of these promoters are upstream of structural genes (ctaD and coxII) for individual subunits of the cytochrome aa3 respiratory complex. The third promoter is that for the cycFG operon, which encodes two c-type cytochromes of unknown function, cytochrome c554 and CycG. Primer extension analysis identified a single oxygen-responsive transcription start site for each gene. Utilizing operon fusions to Escherichia coli lacZ as a measure of promoter activity, transcription from the ctaD, coxII and cycFG promoters was approximately twofold higher when cells were grown at high (30%) oxygen tensions than under low (2%) oxygen or anaerobic (photosynthetic) conditions. Analysis of promoter function using specific host mutations indicated that loss of the R. sphaeroides FNR homologue, FnrL, causes a small, but reproducible, increase in cycFG and coxII transcription when cells are grown at 2% oxygen. However, neither the delta FnrL mutation nor alterations in sequences related to a consensus target site for the E. coli FNR protein increased function of any of these three promoters to that seen under aerobic conditions in wild-type cells. From this we conclude that FnrL is not solely responsible for reduced transcription of these three aerobic cytochrome genes under low oxygen or anaerobic conditions. When activity of these three promoters was monitored after cells were shifted from anaerobic (photosynthetic) conditions to a 30% oxygen atmosphere, it took several cell doublings for LacZ levels to increase to those found in steady-state 30% oxygen cultures. From these results, it appears that activity of these promoters is also regulated by a stable molecule whose synthesis or function responds slowly to the presence of high oxygen tensions.

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“…2). In addition, reduced minus oxidized spectra of soluble and membrane extracts from aerobically grown cells which lack CcmA lacked the absorption feature that is present at ϳ550 nm in wild-type cells and that is diagnostic of the c-type cytochromes (data not shown; 4,8,17).…”

mentioning

confidence: 99%

“…To test if the ccmA2 allele caused the lack of c-type cytochromes, the accumulation of these proteins was measured in extracts from aerobically grown cells, a condition where R. sphaeroides contains several c-type cytochromes (4,8,17). The soluble and membrane proteins of wild-type cells that retain peroxidase activity under denaturing conditions (i.e., c-type cytochromes) were not detected in samples prepared from cells lacking CcmA (Fig.…”

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confidence: 99%

Roles for the Rhodobacter sphaeroides CcmA and CcmG Proteins

2001

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Rhodobacter sphaeroides cells containing an in-frame deletion within ccmA lack detectable soluble and membrane-bound c-type cytochromes and are unable to grow under conditions where these proteins are required. Only strains merodiploid for ccmABCDG were found after attempting to generate cells containing either a ccmG null mutation or a ccmA allele that should be polar on to expression of ccmBCDG, suggesting that CcmG has another important role in R. sphaeroides.Because of their vital role in energy generation, there is considerable interest in determining how cytochromes bind their essential heme cofactor. Among cytochromes, c-type cytochromes are unique because heme is covalently attached to two cysteine thiolates of the polypeptide (22). From analyzing a series of genes (ccm) that are required for c-type cytochrome maturation (13, 31), it appears that a c-type cytochrome precursor protein and heme are translocated across the cytoplasmic membrane by the general export pathway and some combination of CcmABCD, respectively. Once exported, the cysteine thiolates at the c-type cytochrome heme attachment site are believed to form an intramolecular disulfide bond that must be reduced prior to covalent heme attachment (13,31). CcmG is proposed to act in a pathway which facilitates the reduction of this disulfide bond because it contains a pair of redox active thiols (26) and has amino acid sequence similarity to disulfide oxidoreductases in the thioredoxin family (13, 31). The fact that some Ccm mutants have defects in assembly of heme a-containing proteins (19) or iron chelator production (9) underscores the importance of defining whether these proteins participate in other processes.To assess the role of Rhodobacter sphaeroides Ccm proteins, we made mutations within the ccmABCDG locus. This facultative phototroph uses c-type cytochromes to generate energy when growing by aerobic respiration, by photosynthesis, or by anaerobic respiration (4,16,17). R. sphaeroides CcmA is involved in c-type cytochrome maturation since a nonpolar mutation in ccmA caused the loss of this class of electron carrier and an inability to grow under conditions when these proteins are required to generate energy. However, CcmG could have other roles in R. sphaeroides since strains containing a polar insertion in ccmG or a mutation in ccmA that should be polar on to expression of ccmBCDG were not isolated.Growth and genetic procedures. R. sphaeroides was grown at 30°C in Sistrom's medium A (3, 28) containing spectinomycin (25 g/ml), kanamycin (25 g/ml), or tetracycline (1 g/ml) when necessary. Sistrom's medium A containing 7.5% sucrose was used to screen for sucrose sensitivity. Escherichia coli was grown at 37°C in Luria-Bertani medium (24) with appropriate combinations of ampicillin (50 g/ml), spectinomycin (25 g/ ml), kanamycin (25 g/ml), tetracycline (10 g/ml), or chloramphenicol (50 g/ml). E. coli DH5␣ (Table 1) was used as a plasmid host while S17-1 was used for plasmid transfer (2). Mating pairs were plated aerobically on Sistrom's min...

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Organization and expression of the Rhodobacter sphaeroides cycFG operon

Cited by 9 publications

References 53 publications

Features of Rhodobacter sphaeroides CcmFH

Features of Rhodobacter sphaeroides CcmFH

Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides

Roles for the Rhodobacter sphaeroides CcmA and CcmG Proteins

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