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Query: UNIPROT:P06889 (Mol)
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Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co-ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer-membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of eukaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25 degrees C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition simulates, at least partially, a high-temperature environment. Furthermore, temperature-shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo-induced alterations of DNA supercoiling coincide with temperature-induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37 degrees C phenotype when cultured at 25 degrees C; it is non-motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.
Mol Microbiol 1994 Apr
PMID:Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling. 805 44

A 4.6 kb DNA region of the Rhizobium meliloti strain AK631 was found to contain seven open reading frames (ORFs), all oriented in the same direction. The putative gene products of four of these ORFs were highly homologous to UreA, UreB and UreC of Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Canavalia ensiformis. The overall organisation of the DNA region analysed was ORF1, ureA (ORF2), ORF3, ureB (ORF4), ORF5, ORF6 and ureC (ORF7), indicating that the organisation of the urease structural genes in R. meliloti differs from that of other urease genes so far characterized. ORF1 was incomplete; only the 3' end of the coding region was present. The six complete ORFs coded for polypeptides of 11.1 (UreA), 8.9 (ORF3), 10.8 (UreB), 15.0 (ORF5), 13.8 (ORF6) and 60.7 kDa (UreC). No sequence homology to known polypeptides could be detected for the gene products of ORF1, ORF3, ORF5 and ORF6. Using a lacZ fusion and insertional mutagenesis it was shown that the seven ORFs identified were all located in the same transcription unit. For mutational analysis a resistance gene cassette was introduced into each of the complete ORFs resulting in apolar mutations. Mutations in ureA, ureB and ureC, but not in ORF3, ORF5 and ORF6, abolished urease activity in R. meliloti. The determination of hydrogen uptake in these R. meliloti mutants revealed that only ORF6 and ureB are necessary for hydrogen uptake.
Mol Gen Genet 1994 Mar
PMID:A 4.6 kb DNA region of Rhizobium meliloti involved in determining urease and hydrogenase activities carries the structural genes for urease (ureA, ureB, ureC) interrupted by other open reading frames. 812 12

Different genomic DNA samples and primer sequences were evaluated in urease (ure) gene-based PCR assays for rapid identification of Helicobacter pylori. Purified DNA and heated (boiled) cell lysates of bacterial cultures from gastric biopsies were tested with three primer sets for unique internal ureA, ureA+B and ureC sequences. The heated-lysates of H. pylori were quick to prepare but more frequently gave unexpected variable or negative PCR results than assays performed on purified DNA, which were highly specific and reproducible for all three primer sets. Results indicated that sensitivity of the assay was linked to the size of the amplified target region rather than any particular strain feature, with the small 294 bp ureC product providing more accurate assays with heated-lysates of H. pylori. We strongly recommend that negative results in any PCR assay should be checked on purified DNA to exclude the possibility of a false-negative result.
Mol Cell Probes 1993 Dec
PMID:Comparison of urease gene primer sequences for PCR-based amplification assays in identifying the gastric pathogen Helicobacter pylori. 814 75

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a approximately 20-fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomal Notl digest fragments; (iii) cosmids containing Notl sites; and (iv) specific genes. Seven hundred and fifty-one cosmids were mapped to one of three contigs covering > 90% of the chromosome, and are represented by a 68-cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis. All currently known H. pylori genes were mapped, including those for a cytotoxin (vacA), cytotoxin-associated protein (cagA), urease and regulatory functions (ureAb, ureD and ureH), catalase (katA), major and minor flagellins (flaA and flaB), heat-shock (stress) and chaperone proteins (dnaK, htA, hspB (groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26 kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility in H. pylori genome organization.
Mol Microbiol 1994 Feb
PMID:Ordered cosmid library and high-resolution physical-genetic map of Helicobacter pylori strain NCTC11638. 815 75

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient beta-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25% of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.
Plant Mol Biol 1993 Jul
PMID:Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants. 834 3

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.
Mol Microbiol 1993 Jul
PMID:Cloning, expression and sequencing of Helicobacter felis urease genes. 841 83

Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease-negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure-lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen-regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R-like transcriptional regulator that can act at sigma 70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.
Mol Microbiol 1993 Apr
PMID:Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes. 849 92

Helicobacter pylori is considered a primary factor in the pathogenesis of gastric disease, and the successful mucosal colonization is linked to its urease activity. In this study, we assessed the effect of antiulcer agent, ebrotidine, on the in vitro activity of H. pylori urease. The results of assays showed a dose-dependent inhibition of the urease activity. A maximum inhibition (77%) in H. pylori urease activity occurred at 2.1 microM ebrotidine. A known H2-blocker, ranitidine, in a parallel experiment gave a maximal inhibition of 73% at a considerably higher concentration (6.4 microM). The results demonstrate that ebrotidine with its combined acid suppressant and anti-H. pylori activities offers an excellent choice in the treatment of H. pylori associated gastric disease.
Biochem Mol Biol Int 1995 Oct
PMID:Inhibition of Helicobacter pylori urease activity by ebrotidine. 867 7

The Proteus mirabilis and plasmid-encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC-like transcriptional activator. Previously, it was shown that the plasmid-encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea- and UreR-dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid-encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid-encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5 alpha background, as determined by primer-extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR-binding sites within the plasmid-encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid-encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid-encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.
Mol Microbiol 1996 Aug
PMID:Activation of transcription at divergent urea-dependent promoters by the urease gene regulator UreR. 886 86

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 microM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99-100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.
Mol Microbiol 1996 Dec
PMID:Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA). 897 21


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