Xer-cise in Helicobacter pylori: one-step transformation for the construction of markerless gene deletions.
BACKGROUND
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.


METHODS
Markerless deletions were obtained 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.


RESULTS
XerH-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, vacA and HP0366 and removal of the selectable marker at each step. The triple mutant had no growth defect suggesting that multiple difH sites per chromosome can be tolerated without affecting bacterial fitness.


CONCLUSIONS
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.
Ondek Pty Ltd. and Helicobacter pylori Research Laboratory, Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, Discipline of Microbiology & Immunology, The University of Western Australia, M504, L Block, QEII Medical Centre, Nedlands, WA, 6009, Australia.
Aleksandra W
AW
Debowski
Jonathan C
JC
Gauntlett
Hong
H
Li
Tingting
T
Liao
Miriam
M
Sehnal
Hans-Olof
HO
Nilsson
Barry J
BJ
Marshall
Mohammed
M
Benghezal
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D024881:Drug Resistance, Bacterial; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D016480:Helicobacter pylori / Q000235:genetics*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D045522:Recombinases; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; D015500:Chloramphenicol O-Acetyltransferase