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Construction of markerless gene deletions in H. pylori

The latest issue of Helicobacter investigates Xer-cise as a one-step transformation for the construction of markerless gene deletions.

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Xer-cise is an efficient selectable marker removal technique that was first applied in Bacillus subtilis and Escherichia coli for the construction of markerless gene deletions.

Xer-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 Xer-cise in Helicobacter pylori.

The triple mutant had no growth defect
Helicobacter

Dr Mohammed Benghezal and colleagues from Australia obtained markerless deletions by a single natural transformation step of the Xer-cise cassette containing rpsL and cat genes, for streptomycin susceptibility and chloramphenicol resistance respectively, flanked by difH 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 XerH mediated excision of the rpsL-cat cassette, resulting in a markerless deletion.
 
The research team successfully applied XerH-mediated removal of the antibiotic marker in 3 different H. pylori strains to obtain markerless gene deletions at very high efficiencies.

The researchers noted that an unmarked triple deletion mutant was also constructed by sequential deletion of ureA, vacA and HP0366 and removal of the selectable marker at each step.

The team observed that the triple mutant had no growth defect suggesting that multiple difH sites per chromosome can be tolerated without affecting bacterial fitness.
 
Dr Benghezal's team concludes, "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."

Helicobacter 2012: 17(6): 435–443
08 November 2012

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