Proteus mirabilis urease: nucleotide sequence determination and comparison with jack bean urease

Jones, Bradley D.; Mobley, Harry L. T.

doi:10.1128/jb.171.12.6414-6422.1989

Cited by 133 publications

(114 citation statements)

References 34 publications

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“…No precise role can be attributed to the UreC-predicted polypeptide in the absence of similarities between its'amino acid sequence and that of the identified UreD, UreE, or UreF polypeptide of the P. mirabilis urease operon (23,32). These polypeptides are believed to be involved in the processing of nickel ions and/or transport of the urea from the extracellular to the intracellular compartment (32).…”

Section: Resultsmentioning

confidence: 99%

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity

1991

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Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylod was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylorz chromosomal DNA which did not lead to urease activity when introduced into E. coi but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.Helicobacter pylori (previously designated Campylobacter pylori) is a small, curved, gram-negative bacillus found in the stomach of patients with active chronic gastritis and duodenal ulcers. Since its discovery by Warren and Marshall (49) and successful isolation by Marshall et al. in 1984 (30), clinical, histological, and bacteriological investigations have been conducted worldwide in an attempt to determine the role of the bacteria as a causative agent in gastroduodenal diseases. H. pylorn is now recognized as the etiological agent of active chronic gastritis (5), and there is accumulating evidence that the organism contributes to peptic ulceration. Several properties commonly associated with H. pylori are suspected to play a role in the pathogenic process of gastritis as well as ulcer formation. These include adhesion to the gastric epithelium layer (17), a property which correlates with the expression of hemagglutinins (11,35), and adhesion to cell lines (10, 36); the production of proteases capable of degrading mucus glycoproteins (42); and production of cytotoxins (22). Whether or not the genes expressing these traits are harbored by all H. pylori isolates is still unknown. In contrast, the expression of very high urease activity responsible for hydrolysis of urea to ammonia and carbon dioxide has been de...

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Section: Resultsmentioning

confidence: 99%

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity

1991

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“…To determine that this polypeptide was UreE, the purified protein was subjected to N-terminal amino acid analysis. Comparison of the first 10 amino acids of the purified protein (Met-Lys-Lys-Phe-Thr-Gln-Ile-Ile-Asp-Gln) with that of the amino acid sequence of P. mirabilis UreE predicted from the nucleotide sequence (16) weight range, 3,000 to 70,000). The protein eluted at 22 min as measured by the relative A280, corresponding to an apparent molecular size of 36 kDa.…”

Section: Resultsmentioning

confidence: 99%

“…Urease genes of P. mirabilis have been cloned (15) and sequenced (16,28,32), and deletion mutations have been constructed (10,32). In the course of these studies, we observed that clones lacking ureE, ureF, or ureG (accessory genes in the P. mirabilis urease gene cluster) do not express catalytically active urease in unsupplemented medium.…”

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confidence: 99%

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography

et al. 1994

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Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 ,uM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp- Proteus mirabilis is not a common cause of urinary tract infection in the healthy host (31). This organism does, however, infect a high proportion of patients with complicated urinary tracts, that is, those with functional or structural abnormalities or with chronic catheterization (31, 34). In these patients, bladder and renal stone formation is a hallmark of infection with this species and is due to the expression of urease (5). The enzyme hydrolyzes urea to CO2 and NH3, often resulting in elevation of urinary pH (24). Alkalinization of the urine leads to precipitation of Ca2+, Mg2+, and other ions to form carbonate-apatite or struvite stones (5). Ureasenegative mutants, constructed by allelic exchange, are unable to form stones in transurethrally infected mice and are significantly less virulent than the urease-positive parent strains (12,13).Urease is one of only four classes of nickel metalloenzymes which also include hydrogenase, methyl coenzyme M reductase, and carbon monoxide dehydrogenase (8). These divalent cations are required for synthesis of catalytically active urease in bacteria and plants (8). It has been observed that the apourease, synthesized in the absence of nickel, is difficult to activate in vivo by the addition of nickel chloride when protein biosynthesis is inhibited (18,30). This suggests that insertion of nickel ions (Ni2+) into the metallocenter takes place primarily at the time of synthesis of the enzyme subunits, although there is some evidence to the contrary (1). Little is known, however,

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“…The region encoding this set of genes was found to be located immediately downstream of the four genes, ureC, ureD, ureA, and ureB, previously described (16). It consisted of a 3.2-kb fragment having five ORFs which were designated (according to the nomenclature used for K aerogenes [26] and Proteus mirabilis [14]) ureI, ureE, ureF, ureG, and ureH.…”

Section: Materuils and Methodsmentioning

confidence: 99%

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

1992

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Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previousl shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 + 0.39 ,umol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E.coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreHl), and 19,819 (Urel). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coil, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the KiebsieUla aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes),. whereas this homology was restricted to a

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Proteus mirabilis urease: nucleotide sequence determination and comparison with jack bean urease

Cited by 133 publications

References 34 publications

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions

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