溶藻弧菌ZJ-T小RNA srvg23535基因突变株的构建及其功能初探

【背景】小RNA被证实对细菌遗传变异、生长繁殖、致病性等具有重要的调控作用,但是在机会致病菌——溶藻弧菌(Vibrioalginolyticus)中研究较少。本实验室前期通过转录组测序和Northern Blot鉴定得到溶藻弧菌ZJ-T中小RNA srvg23535。【目的】初探小RNA srvg23535对溶藻弧菌生物学特性的影响。【方法】利用同源重组技术构建小RNA srvg23535的缺失突变株,并对野生株和srvg23535突变株的菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及生长代谢等生物学特性进行比较研究。【结果】通过对溶藻弧菌小RNA srvg23535基因缺失突变株的生物学特性分析表明,小RNA srvg23535缺失后,对溶藻弧菌菌落形态、运动性、胞外蛋白酶分泌、H_2O_2和Cu~(2+)的压力感应、铁吸收利用、抗生素抗性以及多数测定的碳源、氮源代谢的影响不显著;但突变株对D-海藻糖(D-trehalose)的代谢减弱,对果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)的代谢增强,并可利用丙氨酸-天冬氨酸(Ala-Asp)为氮源。【结论】小RNA srvg23535参与调控溶藻弧菌对D-海藻糖(D-trehalose)、果胶(Pectin)、丙氨酸-谷氨酰胺(Ala-Gln)和丙氨酸-天冬氨酸(Ala-Asp)的代谢,这将为获取srvg23535调控靶标基因,进而阐明其与靶标基因的相互作用机制奠定基础。

Construction and characterization of the sRNA srvg23535 knock-out mutant of Vibrio alginolyticus ZJ-T

Background] Small non-coding RNAs (sRNAs) play important roles in genetic variation, growth, reproduction, and bacterial pathogenicity, but few studies have been conducted on the opportunistic pathogen of Vibrio alginolyticus. We have identified an sRNA, srvg23535, of V. alginolyticus ZJ-T by RNA-seq and Northern Blot. Objective] We studied the effect of srvg23535 on the biological characteristics of V. alginolyticus. Methods] The srvg23535 knock-out mutant was constructed by homologous recombination. The function of srvg23535 was studied with comparing the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance, and metabolism between the wild type and the mutant strains. Results] The biological characteristics analysis of srvg23535 knock-out mutant showed that the absence of srvg23535 had no significant effect on the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance and the metabolism of most of the tested carbon and nitrogen sources in V. alginolyticus. However, the mutant showed a weaker metabolism of D-trehalose, a stronger metabolism of pectin and alanine-glutamine (Ala-Gln), and accessibility to the utilization of alanine-aspartic acid (Ala-Asp) as the sole nitrogen source. Conclusion] The sRNA srvg23535 is involved in the metabolism regulation of D-trehalose, pectin, Ala-Gln and Ala-Asp with laying the foundation for the target genes screening of srvg23535 and further elucidating the regulation mechanism of srvg23535 to its target genes.