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Two-component systems (TCS) are the prevalent mode for bacteria to sense and respond to changes in their natural habitat. An important protein-cofactor and alternative iron source, sensed by TCS, is haem. In the Gram-positive soil bacterium Corynebacterium glutamicum the TCS HrrSA is crucial for the utilization of haem. Besides HrrSA, a homologous haem-dependent TCS termed ChrSA could be identified. For the analysis of transient gene expression of ChrSA targets, appropriate reporters had to be constructed first. Autofluorescent proteins are valuable tools for the in vivo monitoring of gene expression. However, due to the relatively long half live of most fluorescent proteins, visualization of transient changes remains difficult. SsrA-mediated peptide tagging was used for the construction of destabilized eYFP. The C. glutamicum SsrA tag variants (AAEKSQRDYAASV and -AAV) turned out to be suitable for monitoring dynamic gene expression in C. glutamicum. The respective eYFP variants displayed half-lives of ~22 min (ASV) and ~8 min (AAV). Reporter studies using native eYFP provided strong evidence that ChrSA is the main activator of the divergently located operon hrtBA, encoding for a putative haem ABC transporter, which is required to counteract toxic intracellular accumulation of haem. Furthermore, ChrA acts as a repressor of the homologous response regulator hrrA providing first evidence for a close interplay of the TCS HrrSA and ChrSA. The major focus of this work was to assess the close interplay of HrrSA and ChrSA in haem-dependent signal transduction and to uncover mechanisms enforcing specificity. ChrSA and HrrSA share a high sequence similarity and inherit distinct roles in the control of haemhomeostasis. Both TCS exhibit a high level of cross-talk, counteracted by the phosphatase activity of the sensor kinases HrrS and ChrS, which was shown to be specific for their cognate response regulators. Mutation of a conserved glutamine residue within the phosphatase motif (DxxxQ) of HrrS and ChrS led to a highly increased activation of target gene reporters, confirming the catalytical role of this glutamine residue for phosphatase activity. As the phosphatase motif of HrrS and ChrS is completely identical, further catalytical residues involved in phosphatase reaction were identified. Besides phosphatase activity, pathway specificity can further be enhanced by molecular recognition. Analysis of chimeric proteins of HrrS and ChrS delivered first evidence, that residues forming the interface during phosphatase reaction are located inside the dimerization and histidine phosphotransfer (DHp) domain. Taken together, the results emphasize the importance of phosphatase activity and molecular recognition as crucial mechanisms to ensure pathway specificity of these haemdependent and highly related TCS HrrSA and ChrSA in C. glutamicum.
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Interaction of the two-component systems HrrSA and ChrSA in Corynebacterium glutamicum, Eva Hentschel
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- 2015
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