MOLECULAR ANALYSIS OF THE BVG-REPRESSED UREASE OF BORDETELLA-BRONCHISEPTICA

Bordetella bronchiseptica is a ureolytic mammalian respiratory pathoge n. We have investigated the regulation of urease in B. bronchiseptica and the potential role of this enzyme in eukaryotic invasion and intra cellular survival. Our results indicate urease is a bordetella virulen ce repressed gene. Urease activity in virulent B. bronchiseptica BB786 5 is up-regulated from basal levels by 5g l(-1) magnesium sulphate at 37 degrees C. At 30 degrees C, urease activity remained at basal level s, even in the presence on magnesium sulphate, suggesting a second tem perature dependent mechanism of urease regulation was also operating. Urease was not inducible by 10 mM urea nor up-regulated in nitrogen li miting conditions. To evaluate the role of urease in intracellular inv asion and survival urease-negative mutants of B. bronchiseptica BB7865 and B. bronchiseptica BB7866 were created by transposon mutagenesis, and compared to the urease-positive parental strains in a HeLa cell in vasion 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 sub unit protein of Klebsiella aerogenes, Proteus vulgaris, Helicobacter p ylori and Proteus mirabilis respectively. We also show Bordetella pert ussis to contain sequences homologous with a DNA probe containing the gene encoding UreA of B. bronchiseptica indicating the possible presen ce of cryptic urease genes in this species. (C) 1996 Academic Press Li mited