title: Direct random insertion mutagenesis of Helicobacter pylori.
abstract: Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.
authors.affiliation: Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
authors.forename: Ramon
authors.initials: R
authors.lastname: de Jonge
authors.forename: Dennis
authors.initials: D
authors.lastname: Bakker
authors.forename: Arnoud H M
authors.initials: AH
authors.lastname: van Vliet
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Kuipers
authors.forename: Christina M J E
authors.initials: CM
authors.lastname: Vandenbroucke-Grauls
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Kusters
mesh_terms: D015139:Blotting, Southern; D024881:Drug Resistance, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity; D016254:Mutagenesis, Insertional / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014510:Urease / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D014510:Urease