Construction of Staphylococcus aureus RN6390 hmp gene deletion mutant and analysis of its anti nitric oxide sensitivity

Abstract:

Abstract: [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.

Key words:Keywords: Staphylococcus aureus, flavohaemoglobin (HMP), homologous recombination, biofilm