Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner.
Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.
Pelicic
V
V
INSERM U411, Laboratoire de Microbiologie, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France. pelicic@necker.fr
Morelle
S
S
Lampe
D
D
Nassif
X
X
D002876:Chromosomes, Bacterial; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000235:genetics*; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements; D004269:DNA, Bacterial