负调节基因nsdA在链霉菌中同源性及激活沉默抗生素合成基因簇的研究

nsdA基因是在天蓝色链霉菌中发现的抗生素合成负调控基因。以nsdA基因片段为探针,通过Southern杂交发现nsdA存在于多种链霉菌中。根据天蓝色链霉菌和阿维链霉菌的nsdA序列设计PCR引物,扩增多种链霉菌中nsdA基因并测序。发现在不同链霉菌中nsdA基因的相似性高达77%~100%。其中变铅青链霉菌与天蓝色链霉菌A3(2)的nsdA序列100%一致。变铅青链霉菌通常不合成放线紫红素,中断nsdA获得的突变菌株WQ2能够合成放线紫红素;在WQ2中重新引入野生型nsdA,又失去产抗生素能力。表明nsdA的中断可以激活变铅青链霉菌中沉默的放线紫红素生物合成基因簇的表达;nsdA的广泛存在及其序列高度保守则提示可以尝试用于这些菌种的抗生素高产育种。     

Identification of a gene negatively affecting antibiotic production and morphological differentiation in Streptomyces coelicolor A3(2)

SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene negatively affecting Streptomyces differentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.     

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。     

Deletion of scbA enhances antibiotic production in Streptomyces lividans

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.     

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.