Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes

Mörsdorf, Gerhard; Kaltwasser, Heinrich

doi:10.1016/0378-1097(90)90260-w

Cited by 34 publications

(29 citation statements)

References 28 publications

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“…The amino-terminal protein sequence of the large subunit of K. aerogenes urease (30) confirmed the assignment of the ureC gene to this polypeptide. Furthermore, the gene sequences encoding the K. aerogenes urease subunits are 72, 71, and 58% identical to recently reported sequences encoding urease subunits from P. mirabilis (20), Proteus vulgaris (31), and Helicobacter pylori (3) Complementation, analysis of the urease operon genes. When a region extending from the middle of ureE to the end of ureG was deleted from the urease operon (plasmid pKAU601, Fig.…”

Section: Resultssupporting

confidence: 68%

“…In the bacterial cases, transposon insertion mutants or deletion mutants downstream of the urease. structural genes produced all three urease subunits but possessed little or no urease activity (4,11,13,19,31,32,41). Several of these mutants were defective in nonurease subunit peptides, which may correspond to one or more of the accessory genes described here.…”

Section: Resultsmentioning

confidence: 84%

“…A requirement for accessory genes has also been demonstrated for ureases from soybeans (29), the fungus Aspergillus nidulans (25), Providencia stuartii (32), P. mirabilis (19,41), a urease-positive E. coli (4), K. pneumoniae (13), P. vulgaris (31), and Staphylococcus saprophyticus (11). Genetic studies of soybeans demonstrated the presence of two loci that are distinct from the embryo-specific or ubiquitous urease isozyme structural genes (29).…”

Section: Resultsmentioning

confidence: 99%

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Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation

1990

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A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. Six closely spaced open reading frames were found: ureA (encoding a peptide of 11.1 kilodaltons [kDa]), ureB (11.7-kDa peptide), ureC (60.3-kDa peptide), ureE (17.6-kDa peptide), ureF (25.2-kDa peptide), and ureG (21.9-kDa peptide). Immediately after the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are encoded by ureA, ureB, and ureC based on protein structural studies and sequence homology to jack bean urease. Potential roles for ureE, ureF, and ureG were explored by deleting these accessory genes from the operon. The deletion mutant produced inactive urease, which was partially purified and found to have the same subunit stoichiometry and native size as the active enzyme but which contained no significant levels of nickel. The ic acid-glutamine medium supplemented with 100 ,uM NiSO4 and appropriate antibiotics as previously described (33).DNA sequencing. All restriction enzyme'digestions, end fillings, and other common DNA manipulations, unl,ess otherwise stated, were performed by standard procedures (27,34). Sequencing was carried out on a portion of the urease operon from the upstream SstI site to the downstream HindlIl site of plasmid pKAU17 (33) (Fig. 1). This fragment contains all of the urease operon genes but lacks urease activity due to partial deletion of the upstream promoter region. Portions of the SstI-HindIII fragment were cloned into phage vector M13mp19 (43), and a series of unidirectional deletions was constructed for each strand by using exonuclease III and the method of Henikoff (15). Phage DNA from selected deletion clones was purified (28)

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Section: Resultssupporting

confidence: 68%

Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

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Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation

1990

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“…The requirement for accessory genes has been demonstrated for numerous microbial ureases, including K aerogenes (26), Klebsiella pneumoniae (11), Proteus vulgaris (25), Proteus mirabilis (14, 34), Providencia stuartii (27), a urease-positive E. coli (3), and Staphylococcus saprophyticus (10). The genetic organization of the urease regions of some of these is now well documented.…”

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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“…Urease genes have been isolated from Escherichia coli (7), Proteus mirabilis (20,41), Proteus vulgaris (30), Morganella morganni (15), Klebsiella pneumoniae (12), Klebsiella aerogenes (31), Providencia stuartii (29,32), Staphylococcus saprophyticus (11), Ureaplasma urealyticum (2,3), and Helicobacter pylori (5,24). In the members of the family Enterobacteriaceae, the genetic organization of the urease genes appears to be conserved in all species examined thus far.…”

mentioning

confidence: 99%

Insertional inactivation of an Escherichia coli urease gene by IS3411

Collins

Gutman

1992

J Bacteriol

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Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.

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Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes

Cited by 34 publications

References 28 publications

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation

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

Insertional inactivation of an Escherichia coli urease gene by IS3411

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