Cloning, expression and sequencing of Helicobacter felis urease genes

Abstract

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.

Description of ‘Candidatus Helicobacter heilmannii’ based on DNA sequence analysis of 16S rRNA and urease genes

“…heilmannii'-like isolates (type one or two) showed sequence similarities of~73-76 %, 77-80 %, 78-80 % and 81-83 % with H. pylori, H. felis, H. salomonis and H. bizzozeronii, respectively. In agreement with previous studies, the intergenic spacer regions for H. pylori and H. felis were 3 bp and 9 bp, respectively (Ferrero & Labigne, 1993;Labigne et al, 1991). Five of the cheetah isolates and the isolate from the wild dog also had an intergenic spacer region of 9 bp.…”

Prochlorococcus marinus strain PCC 9511, a picoplanktonic cyanobacterium, synthesizes the smallest urease The GenBank accession number for the sequence determined in this work is AF242489.

“…(Clayton et al, 1990 ;Ferrero & Labigne, 1993 ;Solnick et al, 1994) and heterotrimeric for most other bacterial ureases (Mobley et al, 1995). A dimeric structure has been reported for the Ureaplasma urealyticum urease (Saada & Kahane, 1988).…”

Putative effect ofHelicobacter pyloriand gastritis on gastric acid secretion in cat

“…The plates were incubated in an anaerobic jar with a microaerophilic gas-generating kit for 2 days at 37°C. Bacteria that tested positive for urease and oxidase were identified on the basis of PCR analysis, which showed 16S ribosomal DNA sequences to be Ͼ98% identical to those of H. felis or H. heilmannii (22)(23)(24). In most cases, Helicobacter-like organisms were identified to be H. felis, and H. pylori was not detected in any of these animals before experimental infection.…”

“…After H. pylori infection, rapid urease test, PCR, and culture were used to check gastric infestation according to procedures described elsewhere by ourselves and others (21,23,30). Distinction between H. felis and H. pylori was based, if necessary, on two PCR tests, with primer sequences chosen for amplification on 16S RNA and U3 urease genes as previously described (22,30,45). Briefly, culture and PCR were used as references to distinguish H. pylori from H. felis.…”

“…Gastric biopsy samples were gently ground and cultured on Campylobacter-selective agar medium containing sterile horse blood (5% vol/vol), vancomycin (10 mg/l), Trimetoprim (5 mg/l), polymyxin (2,500 IU/l), and amphotericin B (5 mg/l), as previously described (21,22,32). The plates were incubated in an anaerobic jar with a microaerophilic gas-generating kit for 2 days at 37°C.…”