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

ABSTRACT: 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.

PROVIDER: S-EPMC179675 | BioStudies | 1997-01-01T00:00:00Z

REPOSITORIES: biostudies

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Role of the fnrL gene in photosystem gene expression and photosynthetic growth of Rhodobacter sphaeroides 2.4.1.

Project description:Anoxygenic photosynthetic growth of Rhodobacter sphaeroides 2.4.1 requires a functional fnrL gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the puc operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in puc operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of prr or ppsR can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional fnrL gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode.

1998-01-01 | S-EPMC107050 | BioStudies

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene.

Project description:In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

1995-01-01 | S-EPMC177491 | BioStudies

FnrL regulon in Rhodobacter sphaeroides

Project description:In this study, we performed a ChIP-chip experiment to determine the regulon of FnrL in Rhodobacter sphaeroides. We grew R. sphaeroides under anaerobic photosnthetic conditions, in which FnrL is expected to bind DNA and contol gene expression, and immuno-precipitated FnrL, but also sigma70 and the Beta' subunits of RNA polymerase to determine transcription activity. Overall design: DNA immunoprecipitated with polyclonal antibodies against FnrL, sigma70, or the Beta' subunit of RNA polymerase was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.

2010-07-01 | GSE22027 | GEO

Digging deeper into the PpsR regulon of Rhodobacter sphaeroides

Project description:The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA two-component system and the AppA-PpsR pathway. A PrrA- mutant strain of R. sphaeroides is unable to develop under photosynthetic conditions, but when combined with a ppsR mutation, is able to resume growth. In an effort to better characterize the ppsR regulon and to unravel the relationship between the PrrBA and the AppA-PpsR networks, we compared transcriptome profiles from the wild type, PrrA- and PrrA-PpsR- strains under a permissive, common, growth condition: anaerobic-dark-DMSO. Here is stored the microarray data for the PrrA-PpsR- double mutant strain grown under this condition. Keywords: transcriptome profile Overall design: Rhodobacter sphaeroides PrrA-PpsR- grown under anaerobic dark DMSO conditions. Sparged with 95% N2, 5% CO2 in sistrom minimal medium A supplemented with final concentration 0.1% YE, 0.5% DMSO. The total RNA from three independent cultures of R. sphaeroides PrrA-PpsR- grown under anaerobic-dark-DMSO conditions was used. cDNA synthesis, fragmentation, labeling and hybridization are described elsewhere (Roh et al., 2004).

2008-08-31 | GSE9534 | GEO

FnrL regulon in Rhodobacter sphaeroides

Project description:In this study, we performed a ChIP-chip experiment to determine the regulon of FnrL in Rhodobacter sphaeroides. We grew R. sphaeroides under anaerobic photosnthetic conditions, in which FnrL is expected to bind DNA and contol gene expression, and immuno-precipitated FnrL, but also sigma70 and the Beta' subunits of RNA polymerase to determine transcription activity. DNA immunoprecipitated with polyclonal antibodies against FnrL, sigma70, or the Beta' subunit of RNA polymerase was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.

2010-07-01 | E-GEOD-22027 | ArrayExpress

Digging deeper into the PpsR regulon of Rhodobacter sphaeroides

Project description:The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA two-component system and the AppA-PpsR pathway. A PrrA- mutant strain of R. sphaeroides is unable to develop under photosynthetic conditions, but when combined with a ppsR mutation, is able to resume growth. In an effort to better characterize the ppsR regulon and to unravel the relationship between the PrrBA and the AppA-PpsR networks, we compared transcriptome profiles from the wild type, PrrA- and PrrA-PpsR- strains under a permissive, common, growth condition: anaerobic-dark-DMSO. Here is stored the microarray data for the PrrA-PpsR- double mutant strain grown under this condition. Keywords: transcriptome profile Rhodobacter sphaeroides PrrA-PpsR- grown under anaerobic dark DMSO conditions. Sparged with 95% N2, 5% CO2 in sistrom minimal medium A supplemented with final concentration 0.1% YE, 0.5% DMSO. The total RNA from three independent cultures of R. sphaeroides PrrA-PpsR- grown under anaerobic-dark-DMSO conditions was used. cDNA synthesis, fragmentation, labeling and hybridization are described elsewhere (Roh et al., 2004).

2010-06-20 | E-GEOD-9534 | ArrayExpress

Structural and functional analyses of photosynthetic regulatory genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus.

Project description:Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.

1999-01-01 | S-EPMC93921 | BioStudies

Transcriptomic analysis of aerobic respiratory and anaerobic photosynthetic states in Rhodobacter capsulatus and their modulation by global redox regulators RegA, FnrL and CrtJ.

Project description:Anoxygenicphotosynthetic prokaryotes have simplified photosystems that represent ancient lineages that predate the more complex oxygen evolving photosystems present in cyanobacteria and chloroplasts. These organisms thrive under illuminated anaerobic photosynthetic conditions, but also have the ability to grow under dark aerobic respiratory conditions. This study provides a detailed snapshot of transcription ground states of both dark aerobic and anaerobic photosynthetic growth modes in the purple photosynthetic bacterium Rhodobactercapsulatus. Using 18 biological replicates for aerobic and photosynthetic states, we observed that 1834 genes (53?% of the genome) exhibited altered expression between aerobic and anaerobic growth. In comparison with aerobically grown cells, photosynthetically grown anaerobic cells showed decreased transcription of genes for cobalamin biosynthesis (-45?%), iron transport and homeostasis (-42?%), motility (-32?%), and glycolysis (-34?%). Conversely and more intuitively, the expression of genes involved in carbon fixation (547?%), bacteriochlorophyll biosynthesis (162?%) and carotenogenesis (114?%) were induced. We also analysed the relative contributions of known global redox transcription factors RegA, FnrL and CrtJ in regulating aerobic and anaerobic growth. Approximately 50?% of differentially expressed genes (913 of 1834) were affected by a deletion of RegA, while 33?% (598 out of 1834) were affected by FnrL, and just 7?% (136 out of 1834) by CrtJ. Numerous genes were also shown to be controlled by more than one redox responding regulator.

2017-01-01 | S-EPMC5643017 | BioStudies

The use of chromatin immunoprecipitation to define PpsR binding activity in Rhodobacter sphaeroides 2.4.1.

Project description:The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA(-) mutant is unable to grow under photosynthetic conditions, when a ppsR mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an "extended" PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to R. sphaeroides. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.

2008-01-01 | S-EPMC2566209 | BioStudies

Transcriptome and physiological responses to hydrogen peroxide of the facultatively phototrophic bacterium Rhodobacter sphaeroides.

Project description:The transcriptome responses to hydrogen peroxide, H2O2, of the facultatively phototrophic bacterium Rhodobacter sphaeroides grown under semiaerobic conditions were investigated. At 7 min after the addition of 1 mM H2O2, the expression of approximately 9% of all genes (total, 394) was changed reliably by at least twofold. At 30 min, the number of genes (total, 88) and the magnitude of expression changes were much lower, indicating rapid recovery from stress. Two types of responses were observed: (i) an H2O2 stress response per se and (ii) a shift to high-oxygen metabolism. The former response involved the upregulation of genes for H2O2 detoxification, protein folding and proteolysis, DNA damage repair, iron transport and storage, iron-sulfur cluster repair, and the downregulation of genes for protein translation, motility, and cell wall and lipopolysaccharide synthesis. The shift to high-oxygen metabolism was evident from the differential regulation of genes for aerobic electron transport chain components and the downregulation of tetrapyrrole biosynthesis and photosystem genes. The abundance of photosynthetic complexes was decreased upon prolonged exposure of R. sphaeroides to H2O2, thus confirming the physiological significance of the transcriptome data. The regulatory pathways mediating the shift to high-oxygen metabolism were investigated. They involved the anaerobic activator FnrL and the antirepressor-repressor AppA-PpsR system. The transcription of FnrL-dependent genes was down at 7 min, apparently due to the transient inactivation by H2O2 of the iron-sulfur cluster of FnrL. The transcription of the AppA-PpsR-dependent genes was down at 30 min, apparently due to the significant decrease in appA mRNA.

2005-01-01 | S-EPMC1272974 | BioStudies

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