It appears that the most conserved function of the CtrA and CckA proteins in disparate species is related to motility (Quon et al., 1996; Lang & Beatty, 2002; Miller & Belas, 2006; Brilli et al., 2010; Mercer et al., 2010; Bird & MacKrell, 2011). Unlike C. crescentus, the CckA and CtrA proteins are not essential in regulation of the R. capsulatus cell cycle, but CtrA is required for the proper expression of more than 225 genes (Mercer et al., 2010).
However, it is not known whether phosphorylated or unphosphorylated CtrA is the active form of the protein in this species. Recently, Brilli et al. (2010) analyzed 37 α-proteobacterial genomes and identified orthologs of the 14 genes involved in CtrA-dependent cell cycle regulation in C. crescentus. Their bioinformatic analyses of possible CtrA networks further strengthened some GSI-IX in vivo of the previous work indicating that
CtrA regulation find more and function has a patchwork of conservation in different α-proteobacteria, and they identified a possible chpT ortholog in Rhodobacter. To further understand the CtrA network in R. capsulatus, we have analyzed the motility and RcGTA production phenotypes of strains lacking the putative CtrA regulators sciP and chpT in comparison with ctrA and cckA mutants. We also investigated the effects of CtrA phosphorylation state using a phosphomimetic protein, CtrAD51E, and a version of the protein that is unable to be phosphorylated, CtrAD51A. These CtrA mutants have been used in C. crescentus and Rhodospirillum centenum to study CtrA activities (Domian et al., 1999; Jacobs et al., 1999; Ryan et al., 2002; Siam & Marczynski, 2003; Bird & MacKrell, 2011). The CtrAD51E protein mimics CtrA~P in vivo (Domian et al., 1997; Siam & Marczynski, 2003), and
the CtrAD51A mutant serves as a constitutively selleck screening library unphosphorylated form (Ryan et al., 2002). The experimental strains, plasmids, and PCR primers used for this study are listed in Table 1. Rhodobacter capsulatus was grown at 35 °C in anaerobic photoheterotrophic conditions in complex YPS medium (Wall et al., 1975) or aerobically in RCV medium (Beatty & Gest, 1981) supplemented with appropriate antibiotics when necessary: kanamycin (10 μg mL−1) and tetracycline (0.5 μg mL−1). Escherichia coli was grown in LB medium at 37 °C and supplemented with the appropriate antibiotics when necessary: ampicillin (100 μg mL−1), kanamycin (25 μg mL−1), and tetracycline (10 μg mL−1). The ORFs encoding the predicted ChpT (rcc03000) and SciP (rcc01662) homologs were amplified by PCR from genome of R. capsulatus strain SB1003 using the primers chpT-F and chpT-R, and sciP-F and sciP-R, respectively (Table 1). The amplified products were cloned into pGEM-T-Easy. The genes were disrupted by insertion of a ~1.4-kb SmaI fragment of the kanamycin resistance-encoding KIXX cartridge (Barany, 1985) at specific restriction sites within the ORFs.