Identifying Essential Streptococcus sanguinis Genes Using Genome-Wide Deletion Mutation
Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants.
Key wordsEssential genes Streptococcus sanguinis Fusion PCR Deletion mutation Genome-wide
The authors thank Xiaojing Wang, Yuetan Dou, Jerry Z. Xu, Jenishkumar R Patel, Victoria Stone, My Trinh, Karra Evans, Todd Kitten, Danail Bonchev, and Gregory A. Buck for their contributions in this research. The research is supported by grants R01DE018138 (PX) and R01DE023078 (PX) from the National Institutes of Health.
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