金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

Construction of Staphylococcus aureus RN6390 hmp gene mutant and analysis of NO sensitivity

【目的】构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin,HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide,NO)能力及其在细菌生物被膜形成中的作用。【方法】根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠(SNP)为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。

[Object]To investigate the function of flavohaemoglobin（HMP） in Staphylococcus aureus RN6390 under the nitrification pressure,we constructed the hmp gene deletion mutant of RN6390 strain.[Methods]According to principle of homologous recombination,we obtained the up stream and down stream sequences of hmp gene by PCR using chromosomal DNA of S.aureus RN6390 as template.Antibiotics pressure and alternating temperature culture were applied for mutant strain selection.We verified the clones screened out by genome PCR and real-time PCR quantification.Sodium nitroprusside（SNP）,as nitric oxide（NO） donor,was used for NO resistance evaluation.In addition,we compared the bacteria biofilm formation ability of hmp gene mutant strain with wild type.[Results]We successfully constructed hmp gene mutant strain of S.aureus RN6390.The expression of hmp gene was direct correlate with the concentration of exogenous NO.We found that compared to wild type,the mutant strain was more sensitive to NO and it is prone to form bacteria biofilm.[Conclusions ]The successfully constructed hmp gene deletion mutant of S.aureus provided the possibilities to further investigate the biological function of hmp gene in the resistance of S.aureus to NO from host immune system.