文章摘要
rpoS对鳗弧菌MHK3株Hcp表达和杀菌能力的影响*
The effect of rpoS on expression of Hcp and bactericidal activity inV. anguillarum MHK3
投稿时间:2020-05-04  修订日期:2020-07-05
DOI:
中文关键词: 鳗弧菌  rpoS  Hcp  杀菌
英文关键词: Vibrio anguillarum  rpoS  Hcp  Bactericidal activity
基金项目:
作者单位E-mail
王小鹿 中国海洋大学海洋生命学院 青岛 wxiaolu907@163.com 
李杰 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛  
李贵阳 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛  
唐磊 中国海洋大学海洋生命学院 青岛  
杨慧超 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛  
莫照兰 中国海洋大学海洋生命学院 青岛 mzl@ouc.edu.cn 
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中文摘要:
      本文研究了在鳗弧菌(Vibrio anguillarum)MHK3株中,rpoS(σ38)在表型特征、转录水平和翻译水平对VI型分泌系统(T6SS)中Hcp表达的调控作用,以及对T6SS介导的杀菌能力的影响。通过同源重组法进行基因敲除,得到了MHK3?rpoS突变株。检测结果显示,MHK3?rpoS的生长情况、泳动性、明胶酶活性及酪蛋白酶活性与野生株相比无显著性差异(p>0.05),而在稳定期的菌膜形成能力上有显著上升p<(0.05)。利用pVIK112质粒在鳗弧菌MHK3和MHK3?rpoS中构建了β-半乳糖苷酶lacZ:: hcp1/2融合菌株。采用ONPG法测定lacZ融合菌株的β-半乳糖苷酶的活性。结果显示, MHK3?rpoS的hcp1/2表达量较MHK3有显著上升(p<0.01)。Western-blot显示稳定期的MHK3?rpoS在胞内和胞外Hcp的分泌均有显著升高(p<0.05)。而在细菌拮抗实验发现MHK3?rpoS对大肠杆菌E5的杀菌能力比MHK3下降了约100倍。研究表明,rpoS对稳定期MHK3的菌膜形成能力具有一定的负调控作用,在转录和翻译水平上负调控Hcp的表达,而对MHK3的杀菌能力发挥一定的正调控作用。
英文摘要:
      This study investigated the regulation of rpoS(σ38) to Hcp in the type VI secretory system (T6SS) by on the aspect of phenotypic characteristics, transcriptional and translational levels, and the effect on T6SS-mediated bactericidal ability in the Vibrio anguillarum wild-type MHK3 strain. MHK3?rpoS mutant strain was constructed by homologous recombination. The growth, motility, gelatinase activity and casease activity of MHK3?rpoS showed no significant difference from wild-type strain MHK3. However, the ability of bacterial biofilm formation in MHK3?rpoS increased significantly at the stable phase (p<0.05). The β-galactosidase fusion strains were constructed using pVIK112 plasmid in the MHK3 and MHK3?rpoS of V.anguillarum. The β-galactosidase activity of lacZ fusion strains were determined by ONPG method. The results showed that the hcp1/2 expression of MHK3?rpoS was significantly higher than MHK3(p<0.01). The results of western-blot showed that the translation and secretion of Hcp protein both increased significantly(p<0.05) in the stable phase after deletion of rpoS in MHK3. Compared with MHK3, the bactericidal ability of MHK3?rpoS against E. coli E5 decreased for about 100 fold . The current study indicated that, in MHK3, rpoS was a negative regulator to the bacterial biofilm formation in stable phase and the expression of Hcp at the transcriptional and translation levels. Furthermore, rpoS plays a positive regulatory role in the bactericidal ability of MHK3.
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