title: A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.
abstract: The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Yiqi
authors.initials: Y
authors.lastname: Su
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China.
authors.forename: Huixing
authors.initials: H
authors.lastname: Lin
authors.affiliation: China Animal Health and Epidemiology Center, Qingdao, 266000, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China.
authors.forename: Hongjie
authors.initials: H
authors.lastname: Fan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002681:China; D004251:DNA Transposable Elements; D041722:Genes, Transgenic, Suicide; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D065288:Serogroup; D018502:Streptococcus equi / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D017011:Streptococcus suis / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D013552:Swine; D013696:Temperature; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D037521:Virulence Factors