Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease

Abstract

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).