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

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。

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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 I fragment containing the rpoS gene and its flanking sequence was obtained by screening the genomic DNA library of Pseudomonas sp. M18.A sigma38-subunit-deficient mutant M18S was constructed with insertional gentamycin gene cassette. In PPM medium, the mutant M18S produced 20.4 microg/mL of PCA and 75 microg/mL of Plt. In KMB medium, the mutant M18S produced no PCA and 185.6 microg/mL of Pit. It is obvious that the deficiency of sigma38 subunit in the mutant M18S leads less or no PCA production and much more Plt 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, beta-galactosidase activities of the translational fusions phzA'-'lacZ and pltA'-'lacZ in strain M18S confirmed the effects of sigma38 subunit on PCA and Plt biosynthetic operons. With these results, it is suggested that sigma38 subunit gives a differential impacts on PCA and Plt biosynthesis, i. e, PCA production is positively regulated but Plt production is negatively influenced by sigma38 subunit in Pseudomonas sp. M18.