Open AccessA single‐step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker
Author(s) -
Oswald Winfried,
Tonpitak Walaiporn,
Ohrt Gisela,
Gerlach GeraldF.
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb08721.x
Subject(s) - actinobacillus pleuropneumoniae , biology , microbiology and biotechnology , kanamycin , shuttle vector , serotype , plasmid , genetics , gene , vector (molecular biology) , antibiotics , recombinant dna
Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis , we developed a sucrose‐based counterselection system that allows rapid curing of an Escherichia coli‐A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn 903 kanamycin resistance determinant (km r ) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae . The resultant stable plasmid cointegrates were kanamycin‐resistant, sucrose‐sensitive, and urease‐positive. A simple counterselection on sucrose‐containing agar plates without an additional transconjugation step allowed the efficient isolation of urease‐negative A. pleuropneumoniae mutants that had lost the km r ‐ sacB cassette.