In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site.
BACKGROUND
Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects.


METHODS
A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF).


RESULTS
A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice.


CONCLUSIONS
This complementation strategy should greatly facilitate genetic experiments in H. pylori.
Langford
Melanie L
ML
Biology Department, University of South Alabama, Mobile, AL 36688, USA.
Zabaleta
Jovanny
J
Ochoa
Augusto C
AC
Testerman
Traci L
TL
McGee
David J
DJ
D000818:Animals; D001119:Arginase / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D005816:Genetic Complementation Test; D014644:Genetic Variation; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D001120:Arginine; D001119:Arginase