Generation of a Tn5 transposon library in Haemophilus parasuis and analysis by transposon-directed insertion-site sequencing (TraDIS).
Haemophilus parasuis is an important respiratory tract pathogen of swine and the etiological agent of Glässer's disease. The molecular pathogenesis of H. parasuis is not well studied, mainly due to the lack of efficient tools for genetic manipulation of this bacterium. In this study we describe a Tn5-based random mutagenesis method for use in H. parasuis. A novel chloramphenicol-resistant Tn5 transposome was electroporated into the virulent H. parasuis serovar 5 strain 29755. High transposition efficiency of Tn5, up to 10(4) transformants/μg of transposon DNA, was obtained by modification of the Tn5 DNA in the H. parasuis strain HS071 and establishment of optimal electrotransformation conditions, and a library of approximately 10,500 mutants was constructed. Analysis of the library using transposon-directed insertion-site sequencing (TraDIS) revealed that the insertion of Tn5 was evenly distributed throughout the genome. 10,001 individual mutants were identified, with 1561 genes being disrupted (69.4% of the genome). This newly-developed, efficient mutagenesis approach will be a powerful tool for genetic manipulation of H. parasuis in order to study its physiology and pathogenesis.
Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. sl470@cam.ac.uk
Shi-Lu
SL
Luan
Roy R
RR
Chaudhuri
Sarah E
SE
Peters
Matthew
M
Mayho
Lucy A
LA
Weinert
Sarah A
SA
Crowther
Jinhong
J
Wang
Paul R
PR
Langford
Andrew
A
Rycroft
Brendan W
BW
Wren
Alexander W
AW
Tucker
Duncan J
DJ
Maskell
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements; D018274:Electroporation; D015723:Gene Library; D044137:Haemophilus parasuis / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements
Haemophilus parasuis; Tn5 mutagenesis; TraDIS