睾丸酮丛毛单胞菌teiR基因插入失活突变体构建及降解特性分析半

睾丸酮丛毛单胞菌(Comamonas testosteroni)是革兰氏阴性菌,能够以各种类固醇化合物作为唯一碳源和能源,生物信息学分析发现睾丸酮丛毛单胞菌teiR基因编码蛋白属于LuxR-type转录调控蛋白.利用同源重组原理构建teiR基因插入失活突变体C. mut,初步研究TteiR基因在类同醇代谢途径中的作用,以及对睾丸酮丛毛单胞菌关键降解酶3a-HSD/CR的表达影响.结果发现,teiR基因插入失活突变体C.mut在LB培养基中的生长速率不受影响,突变体C.mut抑制了teiR基因和关键降解酶3a-HSD/CR的表达,同时突变体C. mut丧失了以睾丸酮作为碳源进行生长的能力.结果表明teiR基因调节睾丸酮丛毛单胞菌类因醇代谢,同时teiR基因表达对3a-HSD/CR基因的表达是必需的.图7参14

teiR-disrupted Mutant of Comamonas testosteroni Construction and Its Characteristics in Steroid Biodegradation

Comamonas testosteroni is a Gram-negative bacterium, and has the ability to use various steroids as sole carbon and energy sources. Bioinformatics analysis indicates that teiR encoding protein is a LuxR-type regulator. In this paper, recombinant plasmid pKmut was integrated into C. testosteroni chromosome DNA through homologous recombination by electroporation to generate teiR gene knock-out mutant C. mut. Protein expression levels and its degradation ability were determined by enzyme-linked immunosorbent assay and high performance liquid chromatography, respectively. The growth of teiR-disrupted mutant C. mut was not afeected in LB medium, but it inhibited the 3a-HSD/CR and teiR gene expression. Finally, the mutant lost the ability to use testosterone as carbon and energy sources. Our results confirmed that the teiR gene mediated the steroid metabolism in C. testosteroni and was necessary for the 3a-HSD/CR expression. Fig 7, Ref 14