title: Contraselectable streptomycin susceptibility determinant for genetic manipulation and analysis of Helicobacter pylori.
abstract: Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str(r) allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str(r)] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm(r)] and Str(s)) were each transformed with wild-type genomic DNAs, and Str(r) derivatives were selected. The desired Erm(s) Str(r) isolates were obtained at frequencies that ranged from 17 to 96% among Str(r) transformants, with the Erm(s) yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.
authors.affiliation: Department of Molecular Microbiology, Campus Box 8230, Washington University Medical School, 4940 Parkview Place, St. Louis, MO 63110, USA.
authors.forename: Daiva
authors.initials: D
authors.lastname: Dailidiene
authors.forename: Giedrius
authors.initials: G
authors.lastname: Dailide
authors.forename: Dangeruta
authors.initials: D
authors.lastname: Kersulyte
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Berg
mesh_terms: D000483:Alleles; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005821:Genetic Techniques; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics*; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D013307:Streptomycin / Q000494:pharmacology*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D013307:Streptomycin