title: Identifying essential Streptococcus sanguinis genes using genome-wide deletion mutation.
abstract: 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.
authors: Philips Institute, Virginia Commonwealth University, Richmond, VA, USA.
authors: Lei
authors: L
authors: Chen
authors: Xiuchun
authors: X
authors: Ge
authors: Ping
authors: P
authors: Xu
mesh_terms: D017931:DNA Primers / Q000378:metabolism; D005798:Genes, Bacterial; D020043:Genes, Essential; D017384:Sequence Deletion / Q000235:genetics*; D013298:Streptococcus sanguis / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers
references: J Bacteriol. 2009 Jul;191(13):4166-79
references: 19395487
references: Microbiology. 2009 Aug;155(Pt 8):2573-82
references: 19423626
references: Sci Rep. 2011;1:125
references: 22355642
references: PLoS One. 2012;7(6):e40034
references: 22768210
references: Trends Biotechnol. 2012 Nov;30(11):601-7
references: 22951051
references: J Vis Exp. 2012;(69). pii: 4356. doi: 10.3791/4356
references: 23207952
references: N Engl J Med. 2001 Nov 1;345(18):1318-30
references: 11794152
references: Scand J Dent Res. 1987 Oct;95(5):369-80
references: 3477852
references: Adv Exp Med Biol. 1997;418:1019-21
references: 9331824
references: Infect Immun. 2005 Sep;73(9):6064-74
references: 16113327
references: J Bacteriol. 2007 Apr;189(8):3166-75
references: 17277061
references: Mol Syst Biol. 2008;4:174
references: 18319726