Studies Of Multiple Regulation Role And Mechamism Of Streptomyces. Coelicolor Sigma (T)

In their soil habitat and during their developmental programme, Streptomyces.coelicolor is challenged with diverse nutritional and environmental stresses including high osmolarity, heat or cold shock, pH variation, and nutrient starvation.ECF sigma factors play an important role in the response to these environmental stress.The ECF subfamily is the largest group among theσ70 family and its members are involved in a wide range of environmental responses. The focus of this work is ECF sigma factorσT. The thesis is composed of two parts.First is in regard to the proteolytic pathway of SigT in S. coelicolor mutant rstA, result obtained confirmed the importance of RstA in stabilizing SigT. Second part is in regard to the regulation role of SigT on secondary metabolism of S. coelicolor under amino-acid limited and phosphates limited condition.This work laid the foundation for further research regarding to the role of SigT played in the regulation of secondary metabolism.Generally, anti-sigma factor antagonize the activity of sigma factor,so phenotype of mutant anti-sigma factor is supposed to be opposite to that of sigma factor.However, phenotype of mutant rstA did not reflect the antagonistic characteristics of RstA to SigT.In this study, we confirmed SigT protein in mutant rstA dispeared when detected by Western blotting.These phenomena suggests SigT in⊿rstA mutant suffered to proteolysis.The well known pathway of proteolysis in S. coelicolor is ClpP-dependent proteolytic pathway, which is often associated with SsrA which is responsible for tagging target protein. So,we first knock out the key genes involved in the ClpP proteolytic pathway,clpP and ssrA respectively. To probe the protein expression of SigT, we introduce sigT-egfp gene with the aid of plasmid pIJ8630 into S.coelicolor⊿ssrA⊿rstA and⊿clpP⊿rstA, As a result SigT protein could be detected in S.coelicolor strain⊿ssrA⊿rstA and⊿clpP⊿rstA by Western blotting analysis. These results demonstrate SigT in rstA muant degraded via the SsrA-clpP dependent proteolytic pathway and RstA stabilize sigT protein. The proteolysis mechanism of SigT is analogue to another ECF a factor sigma R. The transcription organization of sigT operon was identified in this work.The result show that rstB is cotranscribed with sigT, while rstA is transcribed solely.5'RACE experiments revealed that two promoters of sigT and rstB lied upstream of rstB gene, also found three promoters of rstA one of which lied in gene interval region of sigT and rstA, the two other of which lied in sigT gene ORF (open reading frame).It is quite unusual that anti-sigma factor is not cotranscribed with its cognate sigma factor with respect to ECFσfactor.RT-PCR analysis and promoter activity assay showed that sigT is induced in a auto regulate manner. This auto regulate mechanism of sigT is common in most ECF sigma factor described to date.Early study suggests SigT play a negative role on differentiation and secondary metabolism in S.coelicolor. Here we further ananlysis the role of sigT in secondary metabolism of S.coelicolor.We found the obvious effect in secondary metabolism of S.coelicolor⊿ sigT under phosphates or nitrogen-limited condition. The enhanced production of Act, Red was observed in mutant sigT under phosphates-limited cultivate condition, while under nitrogen-limited condition the production of Act and Red in mutant sigT is seriously impaired. SigT played the totally different role in controlling of secondary metabolism under different nutritional condition.Promoter activity assay and RT-PCR revealed the upregulation of sigT expression under nitrogen-limition, suggest sigT is induced by nitrogen stress. To our knowledge, it is first example of an ECF sigma factor that is identified to be involved in response to nutritional stress in S.coelicolor.RT-PCR suggested the gene relA and pstS under the control of SigT, the former is the ppGpp synthetase gene and the later is part of the high-affinity phosphate transport system. The phenotype of these mutants is coincident with mutant sigT. In vitro transcription is required to the further affirmation.This work disclosed the degration mechanism of SigT in the⊿rstA genetic background and confirmed RstA is essential for stability of SigT. We also found the role of SigT in the regulation of secondary metabolism in S.coelicolor under nutrient stress for the first time. The evidences present here make significant sense in elaborating the interaction of phosphate and nitrogen regulation in secondary metabolism of S.coelicolor.