Removal of RbaY should result in an increase in selleck inhibitor RbaV-P and therefore allow unregulated inhibition of the cognate σ factor activity by RbaW; our data support this prediction but also

cannot distinguish this from the possibility that RbaV is the controller of output from the pathway, as discussed further below. The absence of RbaW results in the opposite phenotype compared with loss of RbaV or RbaY, supporting the hypothesis that it might act as a negative regulator of a σ factor that initiates transcription of the RcGTA gene cluster. The ~3-fold increase in RcGTA production in the rbaW mutant did not cause a measurable decrease in the viable cell numbers, suggesting the increase is mostly coming from the ~3% subset of the population normally activated for RcGTA production [61] even though this strain showed a population-wide

increase in RcGTA gene expression (Figure 6A). selleck chemicals llc The rbaVW and rbaW mutant phenotypes were not the same, suggesting a dominant effect of the rbaV mutation. Removal of the predicted anti-σ factor, RbaW, led to increased RcGTA gene expression and production only in the presence of a wild type copy of rbaV. The rbaW mutant had no observable differences in stationary phase cell viability or colony morphology, indicating these effects in the rbaVW strain were caused by loss of RbaV. It is not clear why rbaW (pW) maintained elevated RcGTA levels relative to SB1003, but the results with pVW demonstrate a requirement for upstream expression of rbaV for complementing the loss of

rbaW for this phenotype. These data suggest that RbaV is Isotretinoin the determinant positive regulator of RcGTA in this pathway (Figure 8). The in vitro interaction and two-hybrid experiments showed that RbaV does indeed interact with RbaW. Figure 8 Possible models for Rba effects on RcGTA gene expression. Transcript levels of the genes encoding RbaY, RbaV and RbaW are >2-fold lower in the absence of the response regulator CtrA (grey arrow) [8]. The predicted phosphatase RbaY is proposed to activate the STAS domain-containing RbaV (black arrow) by dephosphorylation in response to signal(s) from an unknown sensor kinase(s) (SKs) (grey arrow). There are then two possible scenarios that result in increased RcGTA gene expression. 1. Dephosphorylation of RbaV allows it to activate selleck undetermined intermediaries (X; black arrow) to increase RcGTA gene expression (grey arrow). In this scenario, the predicted kinase RbaW would serve as an inhibitor of RbaV. 2. Dephosphorylation of RbaV allows it to interact with RbaW to relieve inhibition of an unidentified σ factor that promotes transcription of the RcGTA gene cluster (black arrow). Our data support model 1. Studies of RsbV orthologs in Pseudomonas and Vibrio species have demonstrated that the unphosphorylated version of the STAS domain-containing protein was the key regulator of output in those systems [30, 32]. In V.