Cloning, expression and sequencing of Helicobacter felis urease genes

Richard L. Ferrero, Agnès Labigne

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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 cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole 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 plLL205 which were necessary for the expression of an urease‐positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti‐H. felis rabbit serum. One mutant cione 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 caicuiated moiecuiar 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.

Original languageEnglish
Pages (from-to)323-333
Number of pages11
JournalMolecular Microbiology
Volume9
Issue number2
DOIs
Publication statusPublished - 1 Jan 1993
Externally publishedYes

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