表皮葡萄球菌mscL 基因突变株的构建及其生物学功能

目的初步探讨表皮葡萄球菌mscL 基因的生物学功能。方法构建含有壮观霉素抗性基因（spc）的同源重组质粒
pMAD-ΔmscL，电转入表皮葡萄球菌1457，在42 ℃条件下振荡培养，利用蓝白斑和抗生素抗性筛选表皮葡萄球菌mscL基因敲除
突变株（SE1457-ΔmscL）。通过检测低渗胁迫前后突变株和野生株D600值及CFU的变化观察mscL基因对渗透压的调节作用。
采用微量板半定量方法检测不同条件下mscL 敲除突变对细菌生物膜形成的影响。结果成功构建同源重组质粒pMAD-
ΔmscL，经PCR扩增和测序验证获得了表皮葡萄球菌mscL基因敲除突变株,并通过RT-PCR在转录水平上得到进一步验证。处
于对数生长期的mscL突变株在低渗胁迫下的生存能力显著降低，但其生物膜形成能力与野生株相比无显著差异。结论表皮
葡萄球菌mscL基因可在对数生长期参与渗透压的调节，但对生物膜的形成无影响。

Construction and functional analysis of mscL knockout mutant of Staphylococcus epidermidis

Objective To investigate the biological function of mscL gene in S. epidermidis. Methods A plasmid pMAD-ΔmscL
including the upstream and downstream homologous regions of mscL and spectinomycin resistance gene (spc) was constructed
and transformed into S. epidermidis 1457 by electroporation with continuous subculture at 42 ℃ with shaking. The mscL
knockout mutant (SE1457-ΔmscL) was selected by blue-white colony screening and antibiotic resistance. The D600 and numbers
of viable cells were measured in the mutant and parent strains before and after an osmotic downshift of 0.9 M. The effect of the
mscL knockout on biofilm formation was assessed using a semi-quantitative microtiter plate assay. Results The plasmid
pMAD-ΔmscL was constructed and mscL was deleted from the genome of S. epidermidis 1457. The mscL mutant was verified by
PCR of the genomic DNA, direct sequencing and RT-PCR. During the exponential growth phase, the mutant showed
significantly reduced ability to survive osmotic downshock in comparison with the wild-type strain but their capacity to form
biofilm remained similar. Conclusion The mscL gene may be involved in osmoregulation during the logarithmic growth of S.
epidermidis, but it dose not affect biofilm formation of the bacterium.