Both the

cckA and chpT mutants demonstrated a nearly complete loss in RcGTA activity (Fig. 3a). These findings initially suggested that a loss in either ChpT or CckA resulted in a decrease in RcGTA expression, possibly because of the loss of phosphorelay to CtrA. However, western blot analysis of the cultures demonstrated that both cckA and chpT mutants were expressing the RcGTA capsid protein at wild-type levels, but the protein was not detected in the culture supernatants (Fig. 3b). The extracellular levels of the major capsid protein and RcGTA activity were restored to the mutants upon complementation with the plasmid-borne genes. The gene transfer activity of the sciP mutant was lower than wild type (Fig. 3a) but this difference was not statistically different (Table S2). Introduction of the ctrAD51E

allele restored RcGTA expression and increased activity in the ctrA and ctrA/sciP mutants > twofold relative Epigenetics inhibitor to wild type (Fig. 3a). An increase in activity was also observed in both the wild-type (2.4-fold) and sciP mutant (1.6-fold) strains containing ctrAD51E. BTK inhibitors library CtrAD51E increased RcGTA activity and extracellular capsid protein levels slightly in the cckA and chpT mutants (Fig. 3c). The ctrAD51A gene yielded surprising results as all strains expressing this version of CtrA showed a large increase in capsid protein levels inside the cells relative to wild type (Fig. 3d). The wild type and sciP mutant containing CtrAD51E also demonstrated significant increases in RcGTA activity (Fig. 3a). However, unlike the CtrAD51E protein, activities in the ctrA and ctrA/sciP mutants remained very low (Fig. 3a), which agreed with observed low extracellular capsid

levels (Fig. 3d and f). Introduction of the ctrAD51A allele caused an increase in RcGTA activity and extracellular capsid levels in both the cckA and chpT mutants (Fig. 3a and d). Viable cell counts were performed with the different strains on the same cultures used for the gene transfer bioassays and western blots. None of the strains were affected for growth rate and all reached the same approximate cell density at stationary phase as determined by culture turbidity (data not shown). The ctrA/sciP, chpT, and cckA mutations were found to have Methane monooxygenase no significant effect on the number of colony-forming units (Fig. 4). Unexpectedly, the ctrA mutant showed a significant increase (1.6-fold; P < 0.01) in colony-forming units relative to wild type (Fig. 4). Conversely, the sciP mutant was found to have a significant decrease (~0.5 of wild type; P < 0.01) in colony-forming units (Fig. 4). All anova results are available in Table S3. The introduction of the ctrAD51E and ctrAD51A genes had no effect (Fig. S1). Our experiments with R. capsulatus mutant strains lacking putative orthologs of proteins involved in a pathway controlling CtrA activity in C.