A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins.
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.
Fischer
W
W
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany. schmitt@m3401.mpk.med.uni-muenchen.de
Schwan
D
D
Gerland
E
E
Erlenfeld
G E
GE
Odenbreit
S
S
Haas
R
R
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000633:toxicity; D000091243:Ribosomal Protein S9; D013307:Streptomycin / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics
C086513:AlpA protein, E coli; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D014157:Transcription Factors; C074689:VacA protein, Helicobacter pylori; D013307:Streptomycin