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: In contrast to translocon and effector proteins, HrpB2 was efficiently secreted by HrcU cleavage mutants carrying alanine substitutions within the NPTH motif (Fig. 2). For yet unknown reasons, ectopic expression of hrcU under control of the lac promoter in a hrcU deletion mutant background led to increased HrpB2 secretion that was independent of HrcU cleavage. This implies that HrpB2 secretion is controlled by the amounts of HrcU and occurs prior to HrcU cleavage, which is in agreement with the notion that HrpB2 is an early substrate of the T3S system (Fig. 9). We previously reported that HrpB2 interacts with the C-terminal domain of HrcU (Lorenz et al., 2008b). Here, we show that HrpB2 does not stably interact with GST–HrcU deletion derivatives lacking the NPTH motif or carrying a P265G mutation (shown in the context of both GST–HrcU255–357 and GST–HrcU; Fig. 6). In contrast, N264A and P265A mutations in GST–HrcU255–357 did not significantly affect the interaction between HrcUC and HrpB2. It is conceivable that binding of HrpB2 depends on a certain conformation of HrcUC in or around the NPTH motif that is altered in P265G but not in N264A or P265A HrcU mutant derivatives. However, the P265G mutation presumably did not lead to a complete misfolding of HrcU because the interaction with the putative ATPase regulator HrcL and the general T3S chaperone HpaB was not affected. Notably, HrcUP265G did not promote secretion of HrpB2, which is in contrast to the mutant derivatives HrcUN264A and HrcUP265A (Fig. 2). It is therefore possible that the interaction between HrpB2 and HrcUC is required for the efficient secretion of HrpB2 during the early stage of the T3S process, i.e. prior to HrcU cleavage (Fig. 9).
Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.