Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU.
Bottom Line: T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2.The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage.As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.
Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.Show MeSH
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Mentions: The FlhB/YscU homologue HrcU from X. campestris pv. vesicatoria strain 85-10 contains four transmembrane helices and a C-terminal cytoplasmic region that is proteolytically cleaved in both E. coli and X. campestris pv. vesicatoria (Fig. 1A; Lorenz et al., 2008b; Berger et al., 2010). Cleavage of HrcU presumably occurs at the conserved NPTH motif (amino acids 264–267) as was described for HrcU homologues from animal pathogenic bacteria. To study the contribution of the NPTH motif of HrcU to protein cleavage and function, we introduced point mutations that led to an exchange of each amino acid residue of the NPTH motif by alanine respectively. The resulting HrcU mutant derivatives were analysed as C-terminally c-Myc epitope-tagged proteins in E. coli and X. campestris pv. vesicatoria strain 85-10ΔhrcU by immunoblotting. Using a c-Myc epitope-specific antibody, we detected the full-length HrcU-c-Myc, HrcUT266A-c-Myc and HrcUH267A-c-Myc proteins and/or corresponding cleavage products (Fig. 1B). As full-length HrcU-c-Myc was only detectable in E. coli but not in X. campestris pv. vesicatoria, we assume that the proteolytic cleavage of HrcU-c-Myc in X. campestris pv. vesicatoria was nearly complete (Fig. 1B). We detected increased levels of uncleaved HrcUT266A-c-Myc and HrcUH267A-c-Myc when compared with HrcU-c-Myc, suggesting that mutations of amino acids T266 and H267 of HrcU affect the efficiency of the proteolytic cleavage. The C-terminal HrcU cleavage product was not observed for HrcUN264A-c-Myc and only in significantly reduced amounts for HrcUP265A-c-Myc (upon overexposure of the blot; Fig. 1B and C). We also introduced an additional mutation into HrcU that led to an exchange of the proline residue at position 265 by a glycine. Notably, the P265G exchange resulted in a complete loss of detectable HrcU cleavage (Fig. 1C).
Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.