Effect of deletion of Staphylococcus aureus RNAⅢ on γ-hemolysin expression

Abstract

Objective To construct a RNAⅢ gene knockout mutant of Staphylococcus aureus (S. aureus) and evaluate the effect of RNAⅢ on the expression of γ-hemolysin. Methods In order to construct temperature-sensitive S. aureus knock-out vectors, 2 homologous sequences around RNAⅢ were cloned and inserted into pBT2 vector to construct knock-out plasmid pBT2∷RNAⅢ, and then the obtained plasmids RNAⅢ were transferred into S. aureus strain Newman, finally Newman∷ΔRNAⅢ mutant were prepared with homologous recombination. The transcriptional level of γ-hemolysin hlgA/B/C was detected by quantitative real-time PCR (RT-PCR) and the protein levels in the culture supernatants of Newman wild type strain and Newman∷ΔRNAⅢ were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Results Newman∷ΔRNAⅢ mutant was constructed successfully. RT-PCR analysis revealed that the expression of γ-hemolysin in the Newman∷ΔRNAⅢ mutant was significantly decreased as compared to that of Newman wild type strain. Western blotting also showed the transcription level of γ-hemolysin was significantly reduced in the mutant strain. Conclusion The knockout of RNAⅢ has a great effect on the expression of γ-hemolysin in S. aureus.

Keywords

• staphylococcus aureus
• rnaⅲ
• γ-hemolysin
• homologous recombination