A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation |
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Authors: | D Heuermann and R Haas |
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Institution: | (1) Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Spemannstr. 34, D-72076 Tübingen, Germany, DE;(2) Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstr. 9a, D-80336 München, Germany Fax: +49-89-5380584 E-mail: haas@m3401.mpk.med.uni-muenchen.de, DE |
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Abstract: | A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the
minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat
GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10−7 and 4.7 × 10−7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous
replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly,
both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10−5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation
of a recA-deficient H. pylori mutant by the cloned H. pylorirecA
+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.
Received: 22 April 1997 / Accepted: 4 November 1997 |
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Keywords: | Plasmid vector Bacterial conjugation Natural transformation competence Green fluorescent protein Homologous recombination |
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