title: Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori.
abstract: Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3-5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.
authors.affiliation: Department of Microbiology & Immunology, University of Melbourne, Parkville Vic. 3010, Australia; Microbiological Research Unit, Murdoch Childrens Research Institute, Parkville Vic. 3052, Australia. rebecca.gorrell@mcri.edu.au
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Gorrell
authors.forename: Ji
authors.initials: J
authors.lastname: Yang
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Kusters
authors.forename: Arnoud H M
authors.initials: AH
authors.lastname: van Vliet
authors.forename: Roy M
authors.initials: RM
authors.lastname: Robins-Browne
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005819:Genetic Markers; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007612:Kanamycin / Q000494:pharmacology; D016296:Mutagenesis; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005819:Genetic Markers; D007612:Kanamycin; D002701:Chloramphenicol; D004262:DNA Restriction Enzymes