Jiang Peng, Lu Xinzhi, Hou Fangjie, Lin Zhanjun, Yu Wengong
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
Wei Sheng Wu Xue Bao. 2011 Feb;51(2):196-202.
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.
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.
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.
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.
为研究黄素血红蛋白（HMP）在硝化压力下金黄色葡萄球菌RN6390中的功能，我们构建了RN6390菌株的hmp基因缺失突变体。
根据同源重组原理，以金黄色葡萄球菌RN6390的染色体DNA为模板，通过PCR获得hmp基因的上下游序列。采用抗生素压力和变温培养进行突变株筛选。我们通过基因组PCR和实时PCR定量对筛选出的克隆进行验证。使用硝普钠（SNP）作为一氧化氮（NO）供体进行NO抗性评估。此外，我们比较了hmp基因突变株与野生型的细菌生物膜形成能力。
我们成功构建了金黄色葡萄球菌RN6390的hmp基因突变株。hmp基因的表达与外源性NO的浓度直接相关。我们发现，与野生型相比，突变株对NO更敏感，且更容易形成细菌生物膜。
成功构建的金黄色葡萄球菌hmp基因缺失突变体为进一步研究hmp基因在金黄色葡萄球菌抵抗宿主免疫系统NO中的生物学功能提供了可能。
