title: [Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].
abstract: Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis (STM) is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 lambdapir or S17-1 lambdapir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5alpha (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. slin@webmail.hzau.edu.cn
authors.forename: Lin
authors.initials: L
authors.lastname: Shang
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Liangjun
authors.initials: L
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Li
authors.forename: Sihua
authors.initials: S
authors.lastname: Zhang
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.forename: Yaokun
authors.initials: Y
authors.lastname: Li
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Guo
authors.forename: Rui
authors.initials: R
authors.lastname: Zhou
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009268:Nalidixic Acid / Q000494:pharmacology*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D009268:Nalidixic Acid; D027081:DNA Gyrase