A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Abstract

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori.

Generation of Helicobacter pylori Ghosts by PhiX Protein E-Mediated Inactivation and Their Evaluation as Vaccine Candidates

“…Shuttle and suicide plasmids were introduced into H. pylori strains by conjugation or natural transformation as described previously (15). H. pylori transformants or transconjugants carrying the shuttle plasmid pHPC38 were selected on serum plates containing 6 mg of chloramphenicol per liter.…”

Conjugative Transfer of Chromosomally Encoded Antibiotic Resistance from Helicobacter pylori to Campylobacter jejuni

“…Because the streptomycin resistance in H. pylori is mediated by a single point mutation (K43R) in the rpsL gene product (21), the rpsL gene of the recombinant progeny was sequenced using standard procedures. For this purpose, a 346-bp fragment specific for H. pylori rpsL was amplified using the following primers: RpsL-fwd (5Ј-GAA AAG AAA GGA AAA AGG TGG-3Ј) and RpsLrev (5Ј-GCT TTA GTC TTT TTA GTC CCG-3Ј).…”

“…For this purpose, a 346-bp fragment specific for H. pylori rpsL was amplified using the following primers: RpsL-fwd (5Ј-GAA AAG AAA GGA AAA AGG TGG-3Ј) and RpsLrev (5Ј-GCT TTA GTC TTT TTA GTC CCG-3Ј). Chromosomally encoded Str r clones of H. pylori were selected after transformation with the suicide plasmid pEG21, which carries the point mutation in the rpsL gene, and by growing clones on GC agar plates containing 10 g/ml of streptomycin (21,33).…”

Colonization of C57BL/6J and BALB/c Wild-Type and Knockout Mice withHelicobacter pylori: Effect of Vaccination and Implications for Innate and Acquired Immunity

“…To facilitate the quantitative reisolation of SS1 from mouse stomach, we generated a streptomycin-resistant derivative of SS1 by transformation with plasmid pEG21. The plasmid introduces a point mutation into the chromosomal rpsL gene, causing resistance to streptomycin (18). The resulting mutant strain was named SS1S.…”