title: Novel plasmids for gene expression analysis and for genetic manipulation in the gastric pathogen Helicobacter pylori.
abstract: To facilitate gene expression analysis in the human gastric pathogen Helicobacter pylori, we constructed the plasmids pHPLAC-KAN and pHPLAC-CAT containing a promoterless Escherichia coli lacZ gene located upstream from the antibiotic resistance genes aphA-3 or cat, respectively. The suitability of the plasmids for H. pylori mutagenesis and gene expression analysis was evaluated by plasmid integration into the genome of H. pylori strain 1061 by single homologous recombination, using the rpl9 gene encoding ribosomal protein L9 as target. By monitoring beta-galactosidase production from the resulting rpl9::lacZ fusion, it was demonstrated that H. pylori rpl9 displays the classical growth phase-dependent regulation of components of the protein synthesis machinery, as beta-galactosidase production dropped fivefold in the stationary growth phase. The plasmids described in this study extend our methodological repertoire for genetic modification and molecular analysis of H. pylori, and may also be of use for other bacteria, as the resistance cassettes and the lacZ gene are active in the related Campylobacter species.
authors.affiliation: Department of Microbiology and Hygiene, National Reference Center for Helicobacter pylori, Institute of Medical Microbiology and Hygiene, University Hospital of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. ts.bereswill@t-online.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Bereswill
authors.forename: Ruth
authors.initials: R
authors.lastname: Schönenberger
authors.forename: Arnoud H M
authors.initials: AH
authors.lastname: van Vliet
authors.forename: Johannes G
authors.initials: JG
authors.lastname: Kusters
authors.forename: Manfred
authors.initials: M
authors.lastname: Kist
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D007763:Lac Operon; D010957:Plasmids; D011995:Recombination, Genetic; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D013270:Stomach / Q000382:microbiology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012269:Ribosomal Proteins; C049650:ribosomal protein L9; D001616:beta-Galactosidase