RNA聚合酶σ^(38)亚基缺失对假单胞菌抗生物质合成代谢的影响  

Effects of σ^(38)subunit deletion of RNA polymerase on antibiotics biosynthesis in Pseudomonas

作　　者：葛宜和[1] 裴冬丽[2] 张雪洪[3] 许煜泉[3] 

机构地区：[1]鲁东大学生命科学学院,烟台264015 [2]商丘师范学院生物系,商丘476000 [3]上海交通大学生命科学技术学院,上海200240

出　　处：《微生物学报》2006年第6期939-944,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30370041);江苏省高校自然科学研究计划(05KJB180010);鲁东大学科技基金(20063302)~~

摘　　要：设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。With the designed primers, PCR was carried out using the genomic DNA of Pseudomonas sp. M18 as a template and a 378bp DNA fragment of the rpoS gene was amplified. Then, a 3.1kb EcoR Ⅰ-Xho Ⅰfragment containing the rpoS gene and its flanking sequence was obtained by screening the genomic DNA library of Pseudomonas σ^38 -subunlt-deficlent mutant M18S was constructed with insertional gentamycin gene cassette. In PPM sp. M18.σ^ 38 - -subunit-deficient mutant M18S was constructed with insertional gentamycin gene cassette. In PPM medium, the mutant M18S produced 20.4μg/mL of PCA and 75.4μg/mL of Pit. In KMB medium, the mutant M18S produced no PCA and 1 85.6μg/mL of Pit. It is obvious that the deficiency of σ^38 subunit in the mutant M 18S leads less or no PCA production and much more Ph production than those in the wild type strain M18. PCA and Plt production were restored to the levels in wild type strain after complementation with rpoS gene in trans in strain M18S. Moreover, β- galactosidase activities of the translational fusions phzA′-′lacZ and pltA′-′lacZ in strain M18S confirmed the effects of σ^38 subunit on PCA and Pit biosynthetic operons. With these results, it is suggested that σ^38 subunit gives a differential impacts on PCA and Plt biosynthesis, i. e, PCA production is positively regulated but Ph production is negatively influenced by σ^38subunit in Pseudomonas sp. M18.

关 键 词：假单胞菌M18 藤黄绿脓菌素 吩嗪-1-羧酸 σ^-38 亚基 缺失 

分 类 号：S476[农业科学—农业昆虫与害虫防治]