Molecular analysis of the bvg-repressed urease of Bordetella bronchiseptica

Abstract

Bordetella bronchisepticais a ureolytic mammalian respiratory pathogen. We have investigated the regulation of urease in B. bronchisepticaand the potential role of this enzyme in eukaryotic invasion and intracellular survival. Our results indicate urease is a bordetella virulence repressedgene. Urease activity in virulent B. bronchisepticaBB7865 is up-regulated from basal levels by 5 g l −1magnesium sulphate at 37°C. At 30°C, urease activity remained at basal levels, even in the presence on magnesium sulphate, suggesting a second temperature dependent mechanism of urease regulation was also operating. Urease was not inducible by 10 m murea nor up-regulated in nitrogen limiting conditions. To evaluate the role of urease in intracellular invasion and survival urease-negative mutants of B. bronchisepticaBB7865 and B. bronchisepticaBB7866 were created by transposon mutagenesis, and compared to the urease-positive parental strains in a HeLa cell invasion assay. We demonstrate that increasing the concentration of urea in the assay increased survival of the urease-positive but not urease-negative strains after 24 h, suggesting that urease does have a role in intracellular survival. Partial DNA sequence analysis of an 11.0 kb EcoRI DNA fragment encoding urease activity revealed an open reading frame containing 50%, 45%, 45% and 41% homology to the UreA urease subunit protein of Klebsiella aerogenes, Proteus vulgaris, Helicobacter pyloriand Proteus mirabilisrespectively. We also show Bordetella pertussisto contain sequences homologous with a DNA probe containing the gene encoding UreA of B. bronchisepticaindicating the possible presence of cryptic urease genes in this species.