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Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA.

ABSTRACT: The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase on R. capsulatus ctrA gene expression. These experiments indicate that ctrA expression is higher when cultures are grown in phototrophic (anaerobic) conditions compared with chemotrophic (aerobic) conditions, and is higher when grown in a minimal medium compared with a rich medium. We used several mutants to investigate possible regulatory pathways, and found that in R. capsulatus ctrA is not autoregulated but is regulated by a quorum-sensing system. The expression of ctrA increased as cell cultures moved through exponential phase and into stationary phase, with high levels of expression persisting long after culture turbidity plateaued. Although this growth phase-dependent pattern of expression was also observed in a quorum-sensing mutant, the magnitude of ctrA expression was about 50% of the wild-type strain at all phases. Furthermore, reduction of phosphate concentration in the growth medium decreased ctrA expression in a culture density-independent manner, whereas reduction of malic acid (carbon source) or ammonium (nitrogen source) concentration had no effect. The regulation of ctrA expression in R. capsulatus appears to require the coordination of multiple pathways involved in detecting a variety of environmental conditions.

SUBMITTER: Leung MM

PROVIDER: S-EPMC3673944 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA.

Leung Molly M MM Brimacombe Cedric A CA Beatty J Thomas JT

Microbiology (Reading, England) 20121115 Pt 1

The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase ...[more]

PMID: 23154973

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Project description:Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA.Horizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

Project description:Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the RhodobacteralesIMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

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Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA.

Publications

Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA.

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