Evaluation of the homologous recombination in Helicobacter pylori.
BACKGROUND
Most strategies for direct mutagenesis of Helicobacter pylori primarily involve genomic DNA cloning which is a time-consuming and expensive technique.


METHODS
To make a gene replacement, we propose a strategy using polymerase chain reaction (PCR) amplicons to allow a double homologous recombination in the genome of H. pylori. Different strains were used to validate this strategy and we describe how the amplicon insertion was made with accuracy. Moreover, we looked for the shortest homologous sequence needed to allow a specific gene replacement in H. pylori without any deletion, insertion or mutation at the recombination site. All of the experiments were performed at the flaA locus, whose gene encodes the major flagellin.


RESULTS
Amplicons bearing 500 or 150 bp flanking regions of flaA on each side (depending on the strain) were sufficient to allow the specific insertion of a 1173 bp chloramphenicol cassette into the genome of H. pylori. The insertion was accurate with no substitutions at the insertion locus.


CONCLUSIONS
This information opens the door to other strategies for mutagenesis used for the identification of virulence factors without deleting genes, which would not be based on a negative screening system. For example, they could be useful in performing protein fusion for a better understanding of the virulence factor's mechanism.
Pyndiah
Slovénie
S
INSERM ESPRI 2004, Laboratoire de Bactériologie, UniversitéVictor Segalen Bordeaux 2, 33076 Bordeaux cedex, France.
Ménard
Armelle
A
Zerbib
Frank
F
Mégraud
Francis
F
D000483:Alleles; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005408:Flagellin / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D005408:Flagellin; C067138:flaA protein, bacteria