Analysis Of Wbl Family Proteins And Functional Characterization Of WhiB3 In Mycobacterium Tuberculosis

During infection, Mycobacterium tuberculosis（Mtb） is in activate state or enter into a latent infection state depending on external environments. In this process, the regulation of various physiological processes is necessary, and transcriptional regulators play an essential role. To investigate the regulatory roles of the transcriptional regulators, this thesis focuses on the Wbl family proteins, and especially the role of WhiB3 protein, and including the following five aspects:1. Development of a detection system for analysis of regulators in Mtb. Based on the repressive effects of a dimeric MogR protein on flaA gene transcription in Listeria monocytogenes, we established a detection system. MogR contains two domains: the N-terminal DNA binding domain and a C-terminal zipper dimerization domain. MogR1-220 without of the dimerization domain binds well to DNA, but it could not block the transcription from a promoter with this sequence. Hence, we gained the reporter plasmid with MogR binding sequence upstream of the lacZ gene, and constructed an expression plasmid with the purpose of overexpressing target proteins appended to the C terminus of the MogR fragment. And then we systematically analyse all pissible transcriptional factors in Mtb. Of the 154 regulators tested, 109 were confirmed to be homodimers and 32 act as monomers. Interestingly, the Wbl family proteins, i.e., WhiB1-WhiB7, were all classified as monomers, which were further confirmed as monomeric proteins by bacterial two-hybrid system.2. Interactions of Wbl family proteins with the principal sigma factor σA. To investigate the regulatory mechanisms of monomeric regulators, bacterial two-hybrid and mycobacterial protein fragment complementation assays were applied. Our results indicated that all the Wbl family proteins except WhiB5 could bind with σA. Further mutational analyses confirmed that the R515 site of σA is important for its interaction with WhiB3, WhiB4, WhiB6 and WhiB7. The interation of WhiB3 with σA was chosen to further characterize the regulatory mechanisms of the Wbl proteins, and the essential sites for the interaction of WhiB3 and Sig A were characterized. For Sig A, K508, P517 and S518 were important for WhiB3 binding, and the interaction with σA were abolished when E81 and E83 sites were mutated in WhiB3. Aligment of the C-terminal of Wbl proteins was carried out by using the Clustal X software, and E81 and E83 sites were conserved in several WhiB proteins.3. Role of Mtb WhiB3 in M. smegmetis（Ms）. Transcription level of whiB3 was activated in the stationary phase and also under hypoxia environment. To characterize the role of whiB3 under stress conditions, WhiB3 from Mtb was over-expressed in Ms. The growth rate, the length of the bacteria and the speed of aggregation of Ms were all changed, and mutation of R515 in σA abolished these effects. To determine the mechanisms of this over-expression effects, two genes associated with ribosome, MsrpsF and MsrpsJ, were chosen for further analysis. qRT-PCR data indicate that these two genes were not regulated by overexpression of WhiB3 after induction at the log phase, but not at the stationary phase. To further validate these results, the expression level of MsrpsF in Ms WT and MsΔMswhi B3 was decteted by qRT-PCR, and the expression of Msrps F was not affected by WhiB3, suggesting that the impact of over-expression of WhiB3 might be have different regulatory effects.4. WhiB3 regulates the MMAR₃796-MMAR₃800 operon. The product of pigment, the lysis of red blood cells（RBC） and the uptake of ethidium bromide（EB） were all changed inΔwhiB3 in M. marinum（Mm）, while this effect was complemented after consititutive expression of WhiB3ΔwhiB3 in Mm. Since the production of pigments is related to the synthesis of lipids, five genes related to lipids synthesis were analyzed to screen the regulatory targets. Among these genes, MMAR₃796 was regulated by WhiB3 and WhiB3 could control the transcription of the MMAR₃796-MMAR₃800 operon in the stationary phase.5. Characterization of transcriptional activation mechanism of whi B3 under low pH. Specific binding of Pho P to the promoter of whi B3 in vitro was first confirmed by EMSA, and the regulation of whiB3 by phosphorated Pho P after low pH treatment was then verified in Mm. To further confirm the role of Pho R in the regulation of whiB3, Pho PR and WhiB3 in Mm or Pho PR and WhiB3 in Mtb were complemented in theΔphoPR/ΔMswhiB3 double mutant in Ms. Our data indicated that pho R in Mm could sense the low pH signal and autophosphorylate, which will transfer the signal to Pho P. The activated Pho P will then bind to the promoter region of whi B3 and upregulate the transcription of whiB3.