Characterization of a series of urease-negative transposon mutations of Actinobacillus pleuropneumoniae revealed that many of the mutants had insertions 2 to 4 kbp upstream of the urease gene cluster. A 5-kbp upstream region of DNA was sequenced and found to contain six open reading frames (ORFs) transcribed in the same orientation as the urease genes. As well, a partial ORF, apuR, 202 bp upstream of these six ORFs, is transcribed in the opposite orientation. The predicted product of this partial ORF shows homology with many members of the LysR family of transcriptional regulators. Five of the ORFs (cbiKLMQO) appear to form an operon encoding a putative nickel and cobalt periplasmic permease system. The cbiM and cbiQ genes encode proteins that have sequence similarity with known cobalt transport membrane proteins, and the cbiO gene encodes a cobalt transport ATP-binding protein homologue. The product of the cbiK gene is predicted to be the periplasmic-binding-protein component of the system, though it does not show any sequence similarity with CbiN, the cobalt-binding periplasmic protein. Escherichia coli clones containing this putative transport operon together with the urease genes of A. pleuropneumoniae were urease positive when grown in unsupplemented Luria-Bertani broth, whereas a clone containing only the minimal urease gene cluster required the addition of high concentrations of NiCl 2 for full urease activity. This result supports the hypothesis that nickel is a substrate for this permease system. The sixth ORF, utp, appears to encode an integral membrane protein which has significant sequence identity with mammalian urea transport proteins, though its function in A. pleuropneumoniae remains to be determined.The genes required for urease activity in various bacterial species typically include those encoding the structural subunits and those encoding the accessory proteins involved in insertion of two nickel ions within the catalytic site (24). In bacteria with urea-inducible gene clusters, the regulatory gene, ureR, is also present (24). Because of the requirement for nickel as a cofactor, genes such as Helicobacter pylori nixA and Alcaligenes eutrophus hoxN that encode nickel uptake systems have also been shown to affect urease activity (23,41).We recently identified the urease gene cluster of the gramnegative swine pathogen Actinobacillus pleuropneumoniae (4). The organization of the A. pleuropneumoniae gene cluster was found to be similar to that of other bacterial ureases, with the first three genes encoding the structural subunits (UreABC), and the accessory proteins (UreEFGD) encoded by four contiguous genes downstream. There was no evidence of a regulatory gene (ureR) upstream of the cluster.In order to further characterize the urease activity of A. pleuropneumoniae, a bank of transposon mutants of strain CM5 Nal r was generated and screened for urease-negative isolates. A large number of insertions in the urease-negative mutants mapped upstream of the urease gene cluster. Therefore, the 5-kbp reg...