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Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia 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 ?-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.

SUBMITTER: Walker KA

PROVIDER: S-EPMC2976465 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica.

Walker Kimberly A KA Obrist Markus W MW Mildiner-Earley Shirly S Miller Virginia L VL

Journal of bacteriology 20100924 22

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 resp ...[more]

PMID: 20870771

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Project description:UnlabelledSecretins are multimeric outer membrane pore-forming proteins found in complex export systems in Gram-negative bacteria. All type III secretion systems (T3SSs) have a secretin, and one of these is the YsaC secretin of the chromosomally encoded Ysa T3SS of Yersinia enterocolitica. In some cases, pilotin proteins, which are outer membrane lipoproteins, are required for their cognate secretins to multimerize and/or localize to the outer membrane. However, if secretin multimers mislocalize to the inner membrane, this can trigger the protective phage shock protein (Psp) stress response. During a screen for mutations that suppress YsaC toxicity to a psp null strain, we isolated several independent mutations predicted to increase expression of the YE3559 gene within the Ysa pathogenicity island. YE3559, which we have named ysaP, is predicted to encode a small outer membrane lipoprotein, and this location was confirmed by membrane fractionation. Elevated ysaP expression increased the steady-state level of YsaC but made it less toxic to a psp null strain, and it also decreased YsaC-dependent induction of psp gene expression. Subsequent experiments showed that YsaP was not required for YsaC multimerization but was required for the multimers to localize to the outer membrane. Consistent with this, a ysaP null mutation compromised protein export by the Ysa T3SS. All these observations suggest that YsaP is the pilotin for the YsaC secretin. This is only the second pilotin to be characterized for Yersinia and one of only a small number of pilotins described for all bacteria.ImportanceSecretins are essential for the virulence of many bacterial pathogens and also play roles in surface attachment, motility, and competence. This has generated considerable interest in understanding how secretins function. However, their fundamental differences from typical outer membrane proteins have raised various questions about secretins, including how they are assembled into outer membrane multimers. Pilotin proteins facilitate the assembly of some secretins, but only a small number of pilotins have been identified, slowing efforts to understand common and distinct features of secretin assembly. This study provides an important advance by identifying a novel member of the pilotin family and also demonstrating a method of pilotin discovery that could be broadly applied.

Project description:A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319-328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated in rscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to the hmwABC locus of Haemophilus influenzae. The hmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320-3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

Dataset Information

Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica.

Publications

Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica.

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