Statistically significant differences in motility diameters were identified by one-way anova in R (Chambers et al., 1993). Viable cell counts were performed on the same cultures used for each paired bioassay and western blot experiment.

Serial dilutions were plated and colony-forming units (CFU) were calculated to determine the number of viable cells for each culture. Each mutant strain was compared with the wild type in three biological replicates. Statistically significant differences in viable cell numbers were identified by one-way anova in R (Chambers et al., 1993). The R. capsulatus SB1003 (Strnad et al., 2010) regulatory gene orthologs discussed in this work are rcc01663 (ctrA), rcc01662 (sciP), rcc03000 (chpT), and rcc01749 (cckA); all four genes are predicted to be transcribed as independent mRNAs based on genomic context (Strnad et al., 2010) and transcriptome data (Mercer et al., 2010). We compared the R. capsulatus phosphatase inhibitor library CtrA, CckA, ChpT, and SciP sequences to the C. crescentus orthologs, and the regions of similarities and the conserved protein domains identified (Marchler-Bauer et al., 2010) are shown in Fig. 1. We made strains with disruptions in the chpT and sciP genes to test whether

these proteins were involved in the regulation of motility and RcGTA production, as found for CtrA and CckA (Lang & Beatty, 2000, 2002). Additionally, we constructed a new cckA mutant because the original R. capsulatus mutant strains (Lang & Beatty, 2000, 2002) retained ~70% of the cckA coding sequence undisrupted before the insertional mutation site (between the HA and REC domains; Fig. 1), possibly allowing for the expression of Selleck Bafetinib a partially functional protein. We also created a ctrA/sciP double mutant. Flagellar motility of the cckA, chpT, sciP, and ctrA/sciP mutants was assayed using soft agar stabs and compared with wild-type strain SB1003 and the ctrA mutant (Fig. 2). Motility in both the chpT and cckA mutants was reduced, but not as severely as for the ctrA and ctrA/sciP strains, while sciP disruption had no observable effect. Complementation in trans of chpT

and cckA restored motility. Wild-type ctrA does restore motility in the ctrA mutant, but neither ctrAD51E nor ctrAD51A were able to restore motility in the Fossariinae ctrA, cckA, or chpT mutants. The ctrAD51E gene was able to partially restore motility in the ctrA/sciP double mutant (Fig. 2e). Tests for significant differences in swimming distances were performed, and all anova results are available in Supporting Information, Table S1. RcGTA gene transfer activity was assayed for the ctrA, cckA, chpT, sciP, and ctrA/sciP mutants (Fig. 3a). This was paired with analyses of RcGTA capsid protein levels in both cell and culture supernatant samples by western blotting (Fig. 3b–f). As expected, the ctrA and ctrA/sciP mutants had no detectable RcGTA activity (Fig. 3a) or capsid protein expression (Fig.