Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity

Abstract

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...

Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

“…Mycobacreriurn leprea (GenBank accession no. U00020), Helicobacrerpylori (Labigne et al, 1991). Escherichia coli (GlmM, Dallas et al, 1993;PGM, Lu & Kleckner, 1994).…”

Gastric juice for the diagnosis of H pylori infection in patients on proton pump inhibitors

“…Forward primer (5'-TG GGACTGATGGCGTGAGGG-3') and reverse primer (5'-AAGGGCGTTTTTAGATTTTT-3') were prepared from the urease gene sequence according to the report of Labigne et al [14] . PCR amplification was carried out in a total volume of 50 μL containing 2 μL of 2 mmol/L dNTPs, 1 μL containing 50 ρmol of primer 1, 1 μL containing 50 ρmol of primer 2 (synthesized by ABI Automatic synthesizer), 1 unit of Taq DNA polymerase (Promega), 5 μL of 10 × PCR reaction buffer, 3 mmol/L of MgCl2, 2 μL of DNA template containing 0.5 ng of extracted DNA and total volume rounded to 50 μL by double distilled water.…”

Molecular typing ofHelicobacter pyloriisolates from asymptomatic, ulcer and gastritis patients by urease gene polymorphism

“…Variation at the ureA locus Genomic DNAs of all strains were digested with Hind III, which was predicted by analysis of the published nucleotide sequences [10,11] to lack a recognition site within the 411 bp internal fragment of the ureA gene of H. pylori. In genomic Southern blots, the ureA-411 probe hybridized to one Hind III fragment in each of the 64 strains.…”

“…They are among four genes which are located in a 4-2 kb DNA region, which confers urease activity when genetically transferred to Campylobacter jejuni, a related species which lacks the enzyme. These four genes have been sequenced from H. pylori strain 85P [11] and their order in the chromosome is ure CDAB. This study examined strains isolated from patients with one of two major disease syndromes (gastroduodenal ulcer and gastritis), and asymptomatic subjects infected by H. pylori.…”

“…Similarly, a 1 7 kb fragment spanning the ureC and ureD genes (hereafter termed 'ureCD-1P7'), and corresponding to nt 521 to 2240 of the sequence of Labigne and co-workers [11], was amplified using the primers 5'-TGGGACTGATGGCGTGAGGG-3' and 5'-ATCATGACATCAG-CGAAGTTAAAAATGG-3' [14]. Amplification was carried out from 50 ng of genomic DNA of H. pylori NCTC 11637T.…”