The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus

Abstract

Several members of the () 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 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 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 We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in 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 Members of the are abundant in ocean and freshwater environments. The conserved GTA produced by many 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 extends the model of CtrA regulation from to a member of the 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.

Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus

“…These findings also suggest that chpT transcription is regulated oppositely by QS and the Crp/Fnr regulators. This is supported by binding site predictions for FnrL [3] that suggest it binds at the promoter of chpT and clpX, which encodes a protease known to cleave CtrA [3,38,39]. Deletion of fnrL strongly increased the expression of chpT but only resulted in minimal changes for clpX ( Figure 1B).…”

Integration of cell cycle signals by multi-PAS domain kinases

“…DivL (Fig 1B) uses its kinase-like domain to bind to the phosphorylated response regulator DivK~P (17), predicted to drive a conformational change within DivL’s PAS domains. This rearrangement in the PAS domains is then propagated as a regulatory signal to CckA’s own PAS domains, regulating CckA catalytic activity (Fig 1B) (17), and previous studies indicate that the PAS domains of the two proteins are sufficient for communication between the two proteins (10, 21). However, it has remained unclear whether DivL directly regulates CckA catalytic activity, and how a multi-PAS domain protein can pass distinct signals to a target kinase.…”

Identification and characterization of a direct activator of a gene transfer agent

“…A seminal study of antibiotic gene transfer by GTAs in in situ marine microcosms, observed frequencies that were orders of magnitude greater than any known mechanism 12 . In the model host, Rhodobacter capsulatus , RcGTAs are under the control of a number of conserved global regulatory systems such as the cell cycle regulator CtrA 13–15 , the quorum-sensing regulator GtaR 16,17 and various phosphorelay components such as DivL and CckA 15,18 , however, all of these regulators affect RcGTA production indirectly and thus the mechanism of activation is unclear.…”