力达霉素产生菌球孢链霉菌C-1027中atrA同源基因的克隆及分析

目的：在天蓝色链霉菌Streptomyces coelicolor A3(2)中多效性调节因子AtrA（AtrA-c）可通过激活放线紫红素途径特异性的调节因子ActII-ORF4的转录来控制放线紫红素的产生。在灰色链霉菌Streptomyces griseus NBRC13350和阿维链霉菌Streptomyces avermitilis MA-4680中也发现了AtrA-c 编码基因（atrA-c）的同源基因,分别影响链霉素和阿维菌素的生物合成。本文目的在于探索球孢链霉菌C-1027（Streptomyces globisporus C-1027）中是否存在AtrA,克隆球孢链霉菌C-1027中atrA基因并进行生物信息学分析,为进一步确定其对力达霉素产生的调控作用及调控机制奠定基础。[方法] 采用在球孢链霉菌C-1027中异源表达AtrA-c,来确定AtrA-c对力达霉素产量的影响;通过Southern blot 分析来判断在球孢链霉菌C-1027 基因组中是否有atrA-c同源基因;PCR扩增方法获得球孢链霉菌C-1027 atrA基因（atrA-gl）并测序;通过多种生物信息学软件来分析atrA-gl及其与旁侧基因的组织结构、对已发现的AtrA蛋白进行同源性比对及亲缘关系分析。[结果]在球孢链霉菌C-1027中异源表达天蓝色链霉菌AtrA-c蛋白,发现其对力达霉素的产量有影响。以atrA-c为探针,通过Southern blot分析显示球孢链霉菌C-1027基因组中存在atrA-c的同源基因。PCR扩增得到球孢链霉菌C-1027 的atrA基因的全序列以及该基因上下游的旁侧序列（GenBank/EMBL/DDBJ 登录号GU723707）。通过对球孢链霉菌C-1027、天蓝色链霉菌A3(2)、灰色链霉菌NBRC13350以及阿维链霉菌MA-4680 AtrA蛋白序列进行同源性分析发现,四种AtrA蛋白编码氨基酸序列一致性达到65%至 87%,相似性高达70% 至89%。并且,球孢链霉菌C-1027 atrA基因与相邻基因形成的组织结构与天蓝色链霉菌和灰色链霉菌完全一致。根据蛋白质同源性绘制进化树,发现球孢链霉菌AtrA蛋白与灰色链霉菌AtrA蛋白亲缘关系最近。[结论]确定在球孢链霉菌C-1027中存在atrA同源基因并影响力达霉素的产量,克隆了首个力达霉素生物合成基因簇外的调节基因--atrA基因,通过生物信息学分析初步推测了该基因的功能,为进一步研究AtrA-gl对力达霉素途径特异性级联调控网络的调控关系奠定了基础。     

Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters

The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2 approximately P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2 approximately P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2 approximately P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.     

Increasing the efficiency of heterologous promoters in actinomycetes

An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.     

Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation.

The filamentous soil bacterium Streptomyces coelicolor is known to produce four antibiotics which are genetically and structurally distinct. An extensive search for antibiotic regulatory mutants led to the discovery of absB mutants, which are antibiotic deficient but sporulation proficient. Genetic analysis of the absB mutants has resulted in definition of the absB locus at 5 o'clock on the genetic map. Multiple cloned copies of the actII-ORF4 gene, an activator of synthesis of the antibiotic actinorhodin, restore actinorhodin biosynthetic capability to the absB mutants. These results are interpreted to mean that the failure of absB mutants to produce antibiotics results from decreased expression of the antibiotic genes. The absB gene is proposed to be involved in global regulation of antibiotic synthesis.