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Xer‐cise in H elicobacter pylori : One‐step Transformation for the Construction of Markerless Gene Deletions
Author(s) -
Debowski Aleksandra W.,
Gauntlett Jonathan C.,
Li Hong,
Liao Tingting,
Sehnal Miriam,
Nilsson HansOlof,
Marshall Barry J.,
Benghezal Mohammed
Publication year - 2012
Publication title -
helicobacter
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.206
H-Index - 79
eISSN - 1523-5378
pISSN - 1083-4389
DOI - 10.1111/j.1523-5378.2012.00969.x
Subject(s) - selectable marker , homologous recombination , transformation (genetics) , gene , mutant , biology , genetics , plasmid , insert (composites) , microbiology and biotechnology , mechanical engineering , engineering
Background Xer‐cise is an efficient selectable marker removal technique that was first applied in B acillus subtilis and E scherichia coli for the construction of markerless gene deletions. X er‐cise marker excision takes advantage of the presence of site‐specific Xer recombination in most bacterial species for the resolution of chromosome dimers at the dif site during replication. The identification and functional characterization of the difH /XerH recombination system enabled the development of X er‐cise in H elicobacter pylori . Methods Markerless deletions were obtained by a single natural transformation step of the X er‐cise cassette containing rps L and cat genes, for streptomycin susceptibility and chloramphenicol resistance respectively, flanked by dif H sites and neighboring homologous sequences of the target gene. Insertion/deletion recombinant H . pylori were first selected on chloramphenicol‐containing medium followed by selection on streptomycin‐containing medium for clones that underwent X er H mediated excision of the rps L ‐cat cassette, resulting in a markerless deletion. Results X er H ‐mediated removal of the antibiotic marker was successfully applied in three different H . pylori strains to obtain markerless gene deletions at very high efficiencies. An unmarked triple deletion mutant was also constructed by sequential deletion of ureA , vac A and HP 0366 and removal of the selectable marker at each step. The triple mutant had no growth defect suggesting that multiple dif H sites per chromosome can be tolerated without affecting bacterial fitness. Conclusion Xer‐cise eliminates the need for multiple passages on non selective plates and subsequent screening of clones for loss of the antibiotic cassette by replica plating.