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Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis
Author(s) -
Haas Rainer,
Meyer Thomas F.,
Putten Jos P. M.
Publication year - 1993
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1993.tb01618.x
Subject(s) - biology , transposon mutagenesis , flagellin , shuttle vector , transposable element , plasmid , genetics , gene , insertional mutagenesis , mutant , microbiology and biotechnology , mutagenesis , recombinant dna , vector (molecular biology)
Summary Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin‐resistant H. pylori mutant. The frequency of transformation varied between 5 × 10 −4 and 4 × 10 −6 , depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of Tn Max1 , a mini‐Tn1721 transposon carrying a modified chloramphenicol‐acetyltransferase gene, the cat GC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a cat GC ‐inactivated flaA gene resulted in chloramphenicol‐resistant transformants at an average frequency of 4 × 10 −5 . Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat ‐inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C ‐terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.