Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence.
Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active beta-lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line KatoIII. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.
Odenbreit
S
S
Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany.
Till
M
M
Haas
R
R
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001483:Base Sequence; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005407:Flagella; D005408:Flagellin / Q000235:genetics; D015723:Gene Library; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014169:Transformation, Bacterial; D014407:Tumor Cells, Cultured; D014774:Virulence / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005408:Flagellin; C067138:flaA protein, bacteria; C416532:flaB flagellin