Biological Function Of Mechanosensitive Channel (MscL) And Stress Response Regulator (SigN) In Streptomyces Coelicolor

Streptomyces coelicolor is a representative of soil-dwelling bacteria with a complex lifecycle, which is challenged with diverse nutritional and environmental stresses in its soil habitat and during its developmental process. The genome of S. coelicolor was sequenced in 2002. Abundant documents relate to genome annotation of S. coelicolor. Considering the specificity of various stresses from soil habitat, we selected S. coelicolor as a model of gram-positive bacteria and tried to reveal some properties of some genes about stress response. The thesis is composed of two parts. One part is the biochemical and functional characters of larger conductance mechanosensitive channel in S. coelicolor (Sc-MscL), the other part is the study on a new sigma facor,σ^N, in S. coelicolor.Firstly, we revealed some properties in S. coelicolor under the artifical osmotic stress by chemical microanalysis tools, including automatic amino acid analyst, atomic absortion spectrophotometer and chemical reaction. The results demonstrated NaCl is the best solute for simulation of orginal soil osmotic stress. Secondary metabolism of S. coelicolor is ahead under NaCl and sucrose hyperosmotic stress, and S. coelicolor possess more resisitance to osmotic downshock than that of other soil-dwelling G⁺ bacteria. Furthermore, glutamine and glutamic acid were the main amino acids effusing from the cell under the osmotic downshock. K⁺ and Na⁺ also participated in the response of osmotic downshock regulation. Glycine betaine played a role in low grads osmotic downshock. Based on these results, two stress-related genes were chose as the object of this thesis. One is SCO3190 encoding Sc-MscL, the other is SCO4034 encoding alternative sigma factor,σ^N.In the first part, SCO3190 was aligned with the MscL in Escherichia coli and Mycobacteium tuberculosis suggesting that the SCO3190 encodes the larger conductance mechanosensitive channel, named scmscl corresponding of the protein Sc-MscL. Using the technique of gene knock out, expression of Sc-MscL in E. coli and overexpression of Sc-MscL in S. coelicolor, the physiological functions of Sc-MscL were revealed firstly. The specific regulation to NaCl osmotic stress of Sc-MscL was found by comparing the growth curves of wild-type M145 and scmscl disruptive mutant W2006 under osmotic downshock. Furthermore, abundant spores of W2006 appeared on R₅ plate, which was ahead than that of wild type M145, and there is a little decline about Act synthesis. Subsequently, overexpression the Sc-MscL in S. coelicolor affected the phenotype of colony and Act synthesis. The smaller colony with little goffer produced a mass of blue-pigments related to Act. From the other aspect, Sc-MscL as a membrane protein was overexpressed in E. coli by fusing with GST or His tag. Using in vitro cross-linking, the possible functional pentamer of Sc-MscL was detected suggesting the conserve property of MscL.In the second part, there are 65 kinds of sigma factor in S. coelicolor, among which, mostly sigma fctors are alternative and controlled by their anti-sigma factors. Under stress condition, these alternative sigma factors can activate by developmental or environmental signal and promote the expression of stress-related genes. SigB family includes nine kinds of sigma factors, which are the focus of study of sigma factor in S. coelicolor currently. We selected the one sigN corresponding to SCO4034 as object, which got highest score in alignment of S. coelicolor genome to sigB of Bacillus subtilis. Through sigN knock out, the physiological functions of sigN was illuminated. SigN is involved in differentiation and stress response in S. coelicolor. The sigN knock out mutant strain M145Z default in sporulation with a little aerial mycelium on R₅ plate, and scanning electronic micrographs also revealed no visible sign of sporulation. More importantly, significant declines of Act and Red production were observed in M145Z, which was 41.66% and 32.87% of that of M145, respectively. Production of Act was delayed about four days and Red was ahead about one day. All of the results indicated that SigN was required for proper differentiation and antibiotic synthesis of S. coelicolor. In addition, under six stress conditions including osmotic stress, ethanol stress, oxidative stress, acidic stress, heat shock, cold shock, mutant strain M145Z displayed retarded growth compared to wild type strain M145. Among the stresses, M145Z was more sensitive to oxidative, cold and ethanol stresses than the others, and the growth cycle of M145Z has changed comparing to that of M145, which suggested that the proteins involved in growth cycle were also related to cold, heat and oxidative stress response of SigN in S. coelicolor. However, there was no time change in growth cycle in the condition of osmotic, ethanol and acidic stress between the M145 and M145Z according to the growth curves. These results suggested that there were different pathway of stress response to six stress conditions of SigN.To study the interactive protein of SigN, using in vitro affinity purification followed by ESI-MS/MS, we presented evidences that SigN could interact with ATP synthaseβsubunit (ATPaseβ,SCO5373), which was further confirmed by in vitro pull-down assay in E. coli and fluorescent co-localization assay in mammalian 293 and 3T3 cell lines. Interestingly, luciferase assay showed that sigN deletion increased the intracellular ATP level in S. coelicolor, and given that ATP synthaseβsubunit (ATPaseβ) controls the transformation between ADP and ATP, suggesting that sigma factor SigN is possibly required for regulation of ATP dynamics via interaction with ATPaseβ.Sigma factor regulates the expression of a set of genes. SigN was expressed and purified using the Ni²⁺-chelate affinity beads and combined with the fragments of genome of S. coelicolor. The potential promoter sequences were fished. RT-PCR showed that the maxam transcription of sigN appeared at 72h in R₅ medium. The difference display of proteins were analysed by two-dimensional electrophoresis (2-DE). 22 proteins were identified and involved in differentiation, primary metabolism, antibiotic synthesis and stress response. The result further proved the difference between mutant M145Z and wild type M145 on the phenotype, secondary metabolism, and stress response.