[Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].
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.
Shang
Lin
L
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. slin@webmail.hzau.edu.cn
Li
Wei
W
Li
Liangjun
L
Li
Lu
L
Zhang
Sihua
S
Li
Tingting
T
Li
Yaokun
Y
Liu
Lei
L
Guo
Zhiwei
Z
Zhou
Rui
R
Chen
Huanchun
H
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
D001426:Bacterial Proteins; D009268:Nalidixic Acid; D027081:DNA Gyrase