摘要
天蓝色链霉菌(Streptomyces coelicolor)属于革兰氏阳性细菌，由于具有复杂的形态发育和生理分化过程，同时能产生丰富的次生代谢产物，已成为研究链霉菌的模式菌株，其基因组已于2002年测序完成。天蓝色链霉菌作为一种典型的土壤细菌，在生长和生理代谢中常受到环境胁迫作用，长期进化形成了一套完整的胁迫调控机制。对这些胁迫调控机制的深入研究，有助于我们更加清晰地理解微生物与人类和谐共存的意义，以及更好地将这些微生物为我所用。本课题选择了典型的胁迫调控离子通道蛋白(Larger conductance Mechanosensitive channel，MscL)和Sigma factor(σ~N)为研究对象进行阐述，为深入剖析复杂的环境胁迫调控网络体系提供基础。
首先选取实验室条件下的渗透压改变作为一种模式胁迫条件，对天蓝色链霉菌生长相关数据进行分析，包括高渗胁迫对菌体次级代谢的影响、低渗胁迫对菌体生长的影响、不同溶质造成的渗透压改变对菌体产生的不同影响以及细胞相容性物质(游离氨基酸、离子、甜菜碱)的变化。试验结果表明，NaCl是实验室最佳模拟渗透压改变的溶质、高渗胁迫会造成菌体次级代谢的提前；低渗胁迫对菌体造成的影响经24h基本可以恢复到正常水平，低渗胁迫梯度越大，对菌体的生长抑制也越大，细胞相容性物质中参与低渗胁迫的物质主要有Na~+、K~+，谷氨酰胺和甜菜碱。基于上述研究，对渗透压胁迫的调控因子MscL和σ~N分别进行研究。
通过与大肠杆菌大电导率机械敏感性离子通道(Ec-MscL)编码基因进行序列比对，发现天蓝色链霉菌中存在同源性较高的基因SCO3190，推测其编码大电导率机械敏感性离子通道，并将其编码蛋白命名为Sc-MscL。通过scmscl敲除、体内与体外分别表达Sc-MscL对Sc-MscL的功能进行了研究，结果表明Sc-MscL能专一性应对NaCl产生的低渗胁迫反应，Sc-MscL缺失造成菌体孢子产生提前，且放线菌紫素(blue pigmented polyketide actinorhodin，Act)的产量降低；体内过量表达Sc-MscL致使菌落形态变小、皱褶减少，且Act产量增加。利用大肠杆菌成功表达了GST和His融合的膜蛋白Sc-MscL，通过凝血酶酶切获得单一的Sc-MscL蛋白；体外Cross-linking试验证明，Sc-MscL和其他的菌株同源蛋白一样呈现出保守的五聚体结构。这是首次对天蓝色链霉菌离子通道MscL的生化特性和生理功能进行研究，取得了初步研究成果。
另一方面，基因组信息分析显示天蓝色链霉菌内存在大量的σ因子，这些σ因子大多是可选择性的，即在特定的条件下开启，诱导相关基因的表达。其中，σ~B家族是比较重要的σ因子。本论文研究结果表明，σ~B家族中的σ~N与天蓝色链霉菌的气生菌丝、孢子产生和抗生素合成密切相关，σ~N敲除后菌体在胁迫条件下的生长(盐、酸、氧化、乙醇、冷激、热激)受到不同程度的抑制，且在氧化胁迫、酸胁迫和冷激条件下，菌体的生长周期相对于野生型的生长周期发生了改变。通过体外亲和纯化、pull-down和荧光共定位分析，证明σ~N与ATP合成酶的β亚基(ATPaseβ)存在特异性相互作用，分析σ~N缺失突变株发现胞内ATP水平明显升高，这是首次发现σ因子和能量系统调节相关；通过体外蛋白-DNA结合试验和双向电泳分析了σ~N潜在的调控对象，结果表明编码这些蛋白的基因与形态发育、抗生素合成和胁迫调节相关，从而揭示了σ~N缺失造成表型变化的分子机制。
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 R_5 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 R_5 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 Ni~(2+)-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 R_5 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.
引文
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