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细菌生物膜(bacterial biofilm,BF)与大部分的细菌感染相关,有助于病原菌抵抗外部不利的环境,包括抗生素和抗噬菌体等.为了研究生物膜和抗噬菌体的作用机制,本文以6株抗噬菌体甲型副伤寒杆菌(副甲菌)突变菌作为研究对象,在Rif+(利福平)(200 mg/L)平板中划线并滴加噬菌体验证能否抗噬菌体,将6株突变菌接种于96孔板中,每组3个重复,观察其成膜能力以及生物膜的形态,定点突变和互补实验验证突变菌的噬菌体抗性是否由突变基因所引起.结果显示:划线平板中野生型副甲菌在滴加1.2×106个噬菌体处出现空缺,而6株突变菌在滴加2.4×109个噬菌体后仍能生长,表明6株突变菌具有抗噬菌体特性;6株突变菌中,σ-54依赖的翻译调节器突变菌成膜能力(A595=1.1±0.2)较野生型副甲菌(A595=0.5±0.1)显著性增强,且差异显著(P<0.05),光学显微镜下菌体聚集成粗大的不规则团块;同源重组敲除野生型副甲菌σ-54依赖的翻译调节器,突变菌出现噬菌体抗性,将表达σ-54依赖的翻译调节器的载体转化该突变菌,突变菌又恢复了噬菌体敏感性.结果表明,σ-54依赖的翻译调节器是抗噬菌体和生物膜形成相关的基因.
Bacterial biofilm (BF) is associated with most bacterial infections, which can help pathogens to resist external unfavorable environment, including antibiotics and anti-phage.In order to study the mechanism of action of biofilm and anti-phage, Bacteriophage Paratyphoid A Paramyxovirus was used as the research object and crossed with Rif + (rifampicin) (200 mg / L) plate and added with bacteriophage for bacteriophage identification. Six mutants were inoculated In 96-well plates, three replicates in each group were observed for their ability of membrane formation and biofilm morphology, and site-directed mutagenesis and complementation experiments were performed to verify whether the phage resistance of the mutant was caused by the mutant gene.The results showed that: Paratuberculosis in the dropping of 1.2 × 106 phages at vacancies, and six strains of mutants in the dropping of 2.4 × 109 phage can still grow, indicating that six strains of mutants have anti-phage characteristics; 6 strains of mutants, The membrane-forming ability of sigma-54-dependent translation regulator mutant (A595 = 1.1 ± 0.2) was significantly higher than that of wild-type parathyroid (A595 = 0.5 ± 0.1) Body aggregated into coarse irregular clumps; homologous recombination knockout wild Type ps-54 dependent translation regulator, the bacteriophage appeared bacteriophage resistance, the vector expressing the sigma-54-dependent translation regulator was transformed into the mutant bacterium, and the bacteriophage recovered the phage sensitivity.The results showed that σ The -54-dependent translation regulator is a gene that is resistant to phage and biofilm formation.