title: Efficient construction ofHaemophilus parasuismutants based on natural transformation.
abstract: Studies on virulence factors and pathogenecity ofHaemophilus parasuishave long been hindered by a lack of a consistent system for genetic manipulation. In this study, competence was induced by transferringH. parasuisfrom rich medium to starvation medium media-IV (M-IV) and iscR gene deficient mutants ofH. parasuiswere generated efficiently. Transformation frequency varied from 4.1 × 10-5to 1.1 × 10-8when using circular plasmid, and increased to about 2- to 31-fold when transformed using linearized plasmid. Allele replacement occurred efficiently in 6 strains, which are transformable using both circular and linearized pTRU, but not in another 2 strains which could only be transformed using linearized plasmid. The iscR mutants were stable for at least 20 passagesin vitro.Haemophilus parasuisstrains vary extensively in natural transformation efficiency and the method established here allows for transformation of a larger spectrum of strains with an easily accessed plasmid. This provides important tools for genetic manipulation ofH. parasuis.
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Junxing
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Xiufang
authors.initials: X
authors.lastname: Yuan
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Xu
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Kang
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001431:Bacteriological Techniques; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D044137:Haemophilus parasuis / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D037521:Virulence Factors