Novel Genes Affecting Urease Activity inActinobacillus pleuropneumoniae

Abstract

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...

Comparative and Functional Genomic Analysis of Prokaryotic Nickel and Cobalt Uptake Transporters: Evidence for a Novel Group of ATP-Binding Cassette Transporters

“…In two studies, cbiM, cbiQ, and cbiO genes were identified adjacent to bacte-rial urease genes (7,9) and were shown to be important for the urease activity in cells grown under nickel limitation.…”

Functional characterization ofActinobacillus pleuropneumoniaeurea transport protein, ApUT

“…Examination of the urease gene cluster of the swine pathogen A. pleuropneumoniae (5) resulted in the identification and cloning of a putative urea transporter cDNA encoding a 300-amino acid protein ApUT (GenBank accession no. AAD49729).…”

Genes Encoding Specific Nickel Transport Systems Flank the Chromosomal Urease Locus of Pathogenic Yersiniae

“…For several bacterial species in which nickel-transport systems were characterized, genes specifying these carriers were found within or in proximity to genetic loci encoding the nickel-requiring enzyme urease (1,2,19,26) or hydrogenase (13). In addition, nickel permeases of the ABC family are encoded by gene clusters which are harbored either on a plasmid (10), on a pathogenicity island (26), or adjacent to an insertion sequence (2,26).…”

“…In addition, nickel permeases of the ABC family are encoded by gene clusters which are harbored either on a plasmid (10), on a pathogenicity island (26), or adjacent to an insertion sequence (2,26). In Y. pseudotuberculosis, ureH flanks the yut gene (30), located just downstream of the urease accessory gene ureD, whereas the yntABCDE polycistronic unit is upstream of the ureA structural urease gene.…”

“…For the first time, we demonstrate the presence of two transporters specific for nickel in a prokaryote. The human and animal pathogen Y. pseudotuberculosis possesses (i) an ABC transporter encoded by the yntABCDE gene cluster, which shows some degree of similarity to the Nik system in several gram-negative bacteria (2,18,24,26), and (ii) a single-component carrier encoded by the ureH gene, displaying homology to polypeptides of the nickel-cobalt transporter family exemplified by HoxN (13 (22) and an ABC transporter (17). However, the latter has not been proven to be specific for nickel.…”