Identification of YsrT and Evidence that YsrRST Constitute a Unique Phosphorelay System inYersinia enterocolitica

Abstract

Two-component systems (TCS) and phosphorelay systems are mechanisms used by bacteria and fungi to quickly adapt to environmental changes to produce proteins necessary for survival in new environments. Bacterial pathogens use TCS and phosphorelay systems to regulate genes necessary to establish infection within their hosts, including type III secretion systems (T3SS). The Yersinia enterocolitica ysa T3SS is activated in response to NaCl by YsrS and YsrR, a putative hybrid sensor kinase and a response regulator, respectively. Hybrid TCS consist of a sensor kinase that typically has three well-conserved sites of phosphorylation: autophosphorylation site H1, D1 within a receiver domain, and H2 in the histidine phosphotransferase (HPt) domain. From H2, the phosphoryl group is transferred to D2 on the response regulator. A curious feature of YsrS is that it lacks the terminal HPt domain. We report here the identification of the HPt-containing protein (YsrT) that provides this activity for the Ysr system. YsrT is an 82-residue protein predicted to be cytosolic and α-helical in nature and is encoded by a gene adjacent to ysrS. To demonstrate predicted functions of YsrRST as a phosphorelay system, we introduced alanine substitutions at H1, D1, H2, and D2 and tested the mutant proteins for the ability to activate a ysaE-lacZ reporter. As expected, substitutions at H1, H2, and D2 resulted in a loss of activation of ysaE expression. This indicates an interruption of normal protein function, most likely from loss of phosphorylation. A similar result was expected for D1; however, an intriguing "constitutive on" phenotype was observed. In addition, the unusual feature of a separate HPt domain led us to compare the sequences surrounding the ysrS-ysrT junction in several Yersinia strains. In every strain examined, ysrT is a separate gene, leading to speculation that there is a functional advantage to YsrT being an independent protein.