The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP

Abstract

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Connection Between Chromosomal Location and Function of CtrA Phosphorelay Genes in Alphaproteobacteria

“…On the other hand, establishment of a CtrA phosphorylation gradient might indeed also play a role in Rhodobacterales. The bifunctionality of CckA as a kinase and phosphatase has recently been demonstrated for R. capsulatus ( Farrera-Calderon et al, 2020 ).…”

Persulfide-Responsive Transcription Factor SqrR Regulates Gene Transfer and Biofilm Formation via the Metabolic Modulation of Cyclic di-GMP in Rhodobacter capsulatus

“…It is thought that c-di-GMP affects the CckA-ChpT-CtrA phosphorelay by enhancing the phosphatase activity of CckA, and thereby gene transfer by RcGTA is negatively regulated by nonphosphorylated CtrA [ 21 ]. Indeed, gene transfer activity via RcGTA is affected by altering intracellular c-di-GMP levels by the overexpression of the exogenous c-di-GMP metabolic enzyme [ 21 ], and CckA phosphatase versus kinase activity is modulated by c-di-GMP [ 45 ]. Moreover, in positive regulatory systems of biofilm formation, c-di-GMP allosterically modulates regulators—such as effector kinases in two-component systems or transcription factors—to promote biofilm formation [ 46 , 47 , 48 ].…”

The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit

“…The pleiotropic regulator CtrA is absolutely essential for any detectable RcGTA production, and its phosphorylation state controls the transition from RcGTA assembly and DNA packaging to adornment and lysis (Farrera-Calderon et al, 2021;Lang and Beatty, 2000;Mercer et al, 2010Mercer et al, , 2012Westbye et al, 2013Westbye et al, , 2018. Hence, effective production and release of mature RcGTA particles is dependent on an intact phosphorylation cascade from the response regulator CckA to CtrA via ChpT (Farrera-Calderon et al, 2021;Wang et al, 2014;Westbye et al, 2018). High levels of intracellular c-di-GMP stimulate the phosphatase activity of CckA and help to maintain a higher concentration of unphosphorylated CtrA (Farrera-Calderon et al, 2021;Pallegar et al, 2020aPallegar et al, , 2020b.…”

“…Hence, effective production and release of mature RcGTA particles is dependent on an intact phosphorylation cascade from the response regulator CckA to CtrA via ChpT (Farrera-Calderon et al, 2021;Wang et al, 2014;Westbye et al, 2018). High levels of intracellular c-di-GMP stimulate the phosphatase activity of CckA and help to maintain a higher concentration of unphosphorylated CtrA (Farrera-Calderon et al, 2021;Pallegar et al, 2020aPallegar et al, , 2020b. In its unphosphorylated state, CtrA is required for transcription of gafA (Fogg, 2019).…”

“…Low c-di-GMP levels stimulate CckA kinase activity, leading to CtrA phosphorylation (Farrera-Calderon et al, 2021;Pallegar et al, 2020aPallegar et al, , 2020b. CtrA-P also increases expression of the Per-Arnt-Sim (PAS) domain protein DivL, which further enhances CckA kinase activity (Fogg, 2019;Westbye et al, 2018).…”