Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Gerlach, Gerald-F.

doi:10.1016/0378-1097(88)90259-5

Cited by 7 publications

(10 citation statements)

References 6 publications

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“…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: 83%

“…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: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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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“…Delineation of the number and organization of cloned urease genes has been accomplished by subcloning Sau3A partial digests (Mulrooney and Hausinger, submitted), dele-tion analysis (38,63,98,158,240), Ba/31 digestion (98), and transposon mutagenesis (38,98,158,163,240). Inactivation of urease is easily assayed, and therefore large numbers of mutants can be screened quickly.…”

Section: Bacterial Urease Operonmentioning

confidence: 99%

Microbial ureases: significance, regulation, and molecular characterization.

Mobley¹,

Hausinger²

1989

Microbiological Reviews

845

538

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Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Abstract: Urease is an intracellular, nickel-containing enzyme produced by a variety of plants and bacteriaCorrespondence to:

Cited by 7 publications

References 6 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

Microbial ureases: significance, regulation, and molecular characterization.

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