Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylod was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylorz chromosomal DNA which did not lead to urease activity when introduced into E. coi but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.Helicobacter pylori (previously designated Campylobacter pylori) is a small, curved, gram-negative bacillus found in the stomach of patients with active chronic gastritis and duodenal ulcers. Since its discovery by Warren and Marshall (49) and successful isolation by Marshall et al. in 1984 (30), clinical, histological, and bacteriological investigations have been conducted worldwide in an attempt to determine the role of the bacteria as a causative agent in gastroduodenal diseases. H. pylorn is now recognized as the etiological agent of active chronic gastritis (5), and there is accumulating evidence that the organism contributes to peptic ulceration. Several properties commonly associated with H. pylori are suspected to play a role in the pathogenic process of gastritis as well as ulcer formation. These include adhesion to the gastric epithelium layer (17), a property which correlates with the expression of hemagglutinins (11,35), and adhesion to cell lines (10, 36); the production of proteases capable of degrading mucus glycoproteins (42); and production of cytotoxins (22). Whether or not the genes expressing these traits are harbored by all H. pylori isolates is still unknown. In contrast, the expression of very high urease activity responsible for hydrolysis of urea to ammonia and carbon dioxide has been de...
Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.
Electrophoretic karyotyping, mitochondrial DNA restriction fragment length polymorphism analysis, and PCR amplification of interspersed repeats were used to study the variability, phylogenetic affinities, and biogeographic distribution of wild Saccharomyces cerevisiae enological yeasts. The survey concentrated on 42 individual wine cellars in the Charentes area (Cognac region, France). A limited number (35) of predominant S. cerevisiae strains responsible for the fermentation process have been identified by the above molecular methods of differentiation. One strain (ACI) was found to be distributed over the entire area surveyed. There seemed to be little correlation between geographic location and genetic affinity.
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