Identification of a predicted partner-switching system that affects production of the gene transfer agent RcGTA and stationary phase viability in Rhodobacter capsulatus

Ryan G Mercer,Andrew S Lang

doi:10.1186/1471-2180-14-71

Ryan G Mercer, Andrew S Lang

Open Access

https://doi.org/10.1186/1471-2180-14-71

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Abstract

BackgroundProduction of the gene transfer agent RcGTA in the α-proteobacterium Rhodobacter capsulatus is dependent upon the response regulator protein CtrA. Loss of this regulator has widespread effects on transcription in R. capsulatus, including the dysregulation of numerous genes encoding other predicted regulators. This includes a set of putative components of a partner-switching signaling pathway with sequence homology to the σ-regulating proteins RsbV, RsbW, and RsbY that have been extensively characterized for their role in stress responses in gram-positive bacteria. These R. capsulatus homologues, RbaV, RbaW, and RbaY, have been investigated for their possible role in controlling RcGTA gene expression.ResultsA mutant strain lacking rbaW showed a significant increase in RcGTA gene expression and production. Mutation of rbaV or rbaY led to a decrease in RcGTA gene expression and production, and these mutants also showed decreased viability in the stationary phase and produced unusual colony morphologies. In vitro and in vivo protein interaction assays demonstrated that RbaW and RbaV interact. A combination of gene disruptions and protein-protein interaction assays were unsuccessful in attempts to identify a cognate σ factor, and the genetic data support a model where the RbaV protein that is the determinant regulator of RcGTA gene expression in this system.ConclusionsThese findings provide new information about RcGTA regulation by a putative partner-switching system and further illustrate the integration of RcGTA production into R. capsulatus physiology.

Highlights

Production of the gene transfer agent RcGTA in the α-proteobacterium Rhodobacter capsulatus is dependent upon the response regulator protein CtrA
The finding that loss of CtrA affected expression of R. capsulatus rsbVW homologues, which we propose to rename as rbaVW, prompted us to investigate the role of the RbaV and RbaW proteins, along with another identified Rsb homologue, RbaY, in RcGTA production
R. capsulatus was grown at 35°C under anaerobic photoheterotrophic conditions with YPS medium [44] or aerobically with RCV medium [45] supplemented with appropriate antibiotics when necessary: kanamycin (10 μg ml−1), spectinomycin (20 μg ml−1), and tetracycline (0.5 μg ml−1)

Summary

Introduction

Loss of this regulator has widespread effects on transcription in R. capsulatus, including the dysregulation of numerous genes encoding other predicted regulators. This includes a set of putative components of a partner-switching signaling pathway with sequence homology to the σ-regulating proteins RsbV, RsbW, and RsbY that have been extensively characterized for their role in stress responses in gram-positive bacteria. Production of the R. capsulatus GTA, RcGTA, is regulated through multiple cellular signal transduction systems These include the GtaRI quorum sensing system [3,4] and the phosphorelay proteins CtrA and CckA [5,6] and ChpT [6]. There is no evidence that CtrA acts via direct regulation at the RcGTA promoter to control transcription of these genes and the mechanistic link between CtrA and RcGTA gene expression remains unknown

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