The autoregulatory role of EsaR, a quorum‐sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function

Capsular polysaccharide synthesis and virulence in the plant pathogenic bacterium Pantoea stewartii ssp. stewartii requires the quorum‐sensing regulatory proteins, EsaR and EsaI, and the diffusible inducer N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone. Prior mu‐tational studies suggested that EsaR might function as a repressor of quorum sensing in the control of capsular polysaccharide synthesis. Further, a lux box‐like palindromic sequence coinciding with the putative –10 element of the esaR promoter suggested a possible negative autoregulatory role for EsaR. This report presents genetic evidence that EsaR represses the esaR gene under inducer‐limiting conditions, and that addition of inducer promotes rapid, dose‐dependent derepression. DNA mobility‐shift assays and analyses by surface plasmon resonance refractometry show that EsaR binds target DNAs in a ligand‐free state, and that inducer alters the binding characteristics of EsaR. Physical measurements indicate that the EsaR protein binds N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone, in a 1:1 protein:ligand ratio, and that inducer binding enhances the thermal stability of the EsaR protein. These combined genetic and biochemical data establish that EsaR regulates its own expression by signal‐independent repression and signal‐dependent derepression. Additionally, we provide evidence that EsaR does not govern the expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis.