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PubMed2000

Purification and properties of urease from the leaf of mulberry, Morus alba.

Urease was purified from leaves of mulberry (Morus alba, L.) by ammonium sulfate fractionation, acetone fractionation and sequential column chromatography including Q-Sepharose HP, Phenyl-Sepharose HP, Superdex 200 HR and Mono Q. The enzyme was purified 5700-fold to apparent homogeneity with a recovery of 3.6%. The molecular mass of the enzyme was determined to be 90.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and 175 kDa by gel filtration, indicating that the enzyme was a homodimer. In the western blot analysis, 90.5 kDa subunit of the mulberry leaf urease cross-reacted with antiserum raised against jack bean seed urease. The N-terminal sequence of the first 20 residues of the enzyme revealed that it has a high similarity (80-90%) to ureases from other plant sources, suggesting that the mulberry leaf urease is closely related to other plant ureases. However, the mulberry leaf enzyme showed an optimum pH for activity of 9.0, while the optimum pH of most ureases isolated from plants and bacterial is neutral. In addition, the K(m) value for urea was 0.16 mM, which is lower than those of ureases from other sources. It is also proposed that urease activity ingested by browsing silkworm releases ammonia that is subsequently used in silkworm protein synthesis.

Amino Acid Sequence↗
PubMed2003

Urea permeation and hydrolysis through hollow fiber dialyzer immobilized with urease: storage and operation properties.

The surface of polyacrylonitrile hollow fibers was hydrolyzed and covalently bonded with urease via glutaraldehyde. Immobilized urease retained higher relative activity than native urease when storing at various pHs. The stabilities of immobilized urease to pH were higher than those of native enzyme. Immobilized urease retained 86% of initial activity after reusing 15 times at pH 7. After storing for 42d at 4 degrees C and pH 7, the immobilized urease can hydrolyze 15% of initial concentration of urea at pH 7 and 37 degrees C after 4h, while native urease lost almost its catalytic ability. The removal of urea using urease-immobilized dialyzer was demonstrated with in vitro dialysis and showed faster removing rate of urea than a regular dialyzer by 2 times. Furthermore, the improvement in the urea clearance by the urease immobilization to a dialyzer increased with the dialysate velocity.

Dialysis↗
PubMed2002

Urease activity in microbiologically-induced calcite precipitation.

The role of microbial urease in calcite precipitation was studied utilizing a recombinant Escherichia coli HB101 containing a plasmid, pBU11, that encodes Bacillus pasteurii urease. The calcite precipitation by E. coli HB101 (pBU11) was significant although its precipitation level was not as high as that by B. pasteurii. Addition of low concentrations (5-100 microM) of nickel, the cofactor of urease, to the medium further enhanced calcite precipitation by E. coli (pBU11). Calcite precipitation induced by both B. pasteurii and E. coli (pBU11) was inhibited in the presence of a urease inhibitor, acetohydroxamic acid (AHA). These observations on the recombinant urease have confirmed that urease activity is essential for microbiologically-induced calcite precipitation. Partially purified B. pasteurii urease was immobilized in polyurethane (PU) foam to compare the efficacy of calcite precipitation between the free and immobilized enzymes. The immobilized urease showed higher K(m) and lower V(max) values, which were reflected by a slower overall calcite precipitation. However, scanning electron micrographs (SEM) identified that the calcite precipitation occurred throughout the matrices of polyurethane. Furthermore, PU-immobilized urease retained higher enzymatic activities at high temperatures and in the presence of a high concentration of pronase, indicating that immobilization protects the enzyme activity from environmental changes.

Bacillus↗
PubMed2001

Functional characterisation of urease accessory protein G (ureG) from potato.

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5'-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.

Alleles↗
PubMed2000

Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides.

BACKGROUND & AIMS: The significance of acid-primed recognition of ligands by Helicobacter pylori urease is unknown. This study aimed to further characterize the specificity of urease adherence in vitro and verify whether specific inhibition will translate into in vivo suppression of colonization. METHODS: A highly sensitive competitive enzyme-linked ligand capture assay was used to quantify the capacity of each test inhibitor to compete with labeled mucin for binding sites on immobilized native urease. A model polymer that strongly bound urease was used in an in vivo trial using euthymic hairless mice as an infection model. RESULTS: The blockage of urease-gastric mucin interaction by certain inhibitors revealed an acid-functional lectin-like activity by urease, specifically recognizing bacterial lipopolysaccharides and certain species of polysaccharides, nonbacterial glycolipids, and glycoproteins. Dextran sulfate significantly (P < 0.01) suppressed colonization of mice by H. pylori when given before and/or after challenge. CONCLUSIONS: The acid-driven high-affinity adherence of H. pylori urease to mucin and lipopolysaccharides contributes to gastric mucosal colonization by the bacterium based on in vivo targeting experiments using specific polysaccharides in a mouse model with acute infection. Acid-functional urease-homing polysaccharides that can interfere with urease-mucin or H. pylori whole cell-mucin interaction in vitro can significantly interfere with colonization by the bacterium in vivo.

Acids↗
PubMed1982

Distribution and changes in urease (EC 3.5.1.5) activity in Rumen Simulation Technique (Rusitec).

1. The Rumen Simulation Technique (Rusitec) was used in a series of long-term experiments to study the distribution and changes of urease (EC 3.5.1.5) activity in a heterogeneous fermentation system. 2. It was shown that in Rusitec the high urease activity from the inoculum decreased to low values, that the rate of decrease was consistent with simple dilution of ureolytic micro-organisms and that the urease activity could be restored to original values by infusion of urea into the reaction vessels. The magnitude of this urease activity was a direct function of the amounts of urea infused. Single daily additions of the same or greater amounts of urea in food or as solid failed to increase the urease activity significantly. 3. In general, urease activity increased 2-6 h after feeding and the increases were greater with roughage diets. 4. The ureolytic activity per unit volume was always higher in compartment 2(space occupied by micro-organisms that are loosely associated with the solid) than in compartment 1 (strained rumen contents) or compartment 3 (space occupied by microbial population that cannot be washed out of the solid matrix). 5. The distribution of urease activity between the compartments was different from the distribution of certain other enzymes (e.g. protease and alkaline phosphatase (EC 3.1.3.1)). 6. Apart from the boundary region, the concentrations of urease, ammonia and volatile fatty acids in compartment 2 were constant, while the concentrations of protein, DNA and another enzyme (alkaline phosphatase) increased with the depth of the compartment. Specific urease activity (per unit weight of protein or DNA) was much higher in compartment 1 than in compartment 2 and it decreased markedly with depth of compartment. 7. The concentrations of ammonia were always much higher in the solid matrix (compartments 2 and 3) than in the free suspension of micro-organisms (compartment 1). There was a linear relation between these two quantities. 8. The results are discussed in relation to published work on the entry and metabolism of urea in the rumen.

Alkaline Phosphatase↗
PubMed2000

Admixture with whole blood does not explain false-negative urease tests.

Rapid urease test sensitivity for Helicobacter pylori is reduced in the presence of active upper gastrointestinal bleeding. The aim of this study was to evaluate the in vitro effect of whole blood on rapid urease testing. Urease solution was added to normal saline, and heparinized whole blood both positive and negative for H. pylori antibody. The mixtures were then serially diluted in saline, and/or whole blood and added to three different rapid urease kits. The admixture of urease in H. pylori-seropositive whole blood diluted in either saline or whole blood enhanced performance in both kits fourfold compared with saline alone. No false-negative results were observed in either kit. Seronegative whole blood produced similar results. Undiluted saline or whole blood produced no positive rapid urease tests. Whole blood accelerates the urease reaction in vitro. Neither H. pylori antibody-positive nor -negative whole blood adversely impacted the rapid urease test. False-negative rapid urease test results in upper gastrointestinal bleeding cannot be explained by admixture with whole blood.

Clinical Enzyme Tests↗
PubMed1998

False-negative biopsy urease test in bleeding ulcers caused by the buffering effects of blood.

OBJECTIVES: A false-negative biopsy urease test (BUT) is common in Helicobacter pylori-associated bleeding peptic ulcers. Although blood in the stomach is thought to interfere with the biopsy urease test, the underlying mechanism remains unknown. This in vitro experiment sought to identify the blood component(s) that interfere with the biopsy urease test, and delineate the mechanism of inhibition. METHODS: The modified Hazell's microtiter test was used to detect the urease activity of H. pylori. A positive result was indicated by a color change of the pH indicator, bromothymol blue, at 630 nm. Human whole blood, sera with and without anti-H. pylori antibody, electrolytes, and enzymes were incubated with H. pylori to identify the blood component(s) causing the inhibition of urease activity. In addition, any interference of the pH color indicator was tested by adding different concentrations of serum albumin to the urease reagent that contained a fixed quantity of ammonia in the absence of H. pylori. RESULTS: The color change of the microtiter urease test was significantly reduced by blood (p < 0.0001), regardless of the presence of anti-H. pylori antibody. Electrolytes and serum enzymes did not interfere with the urease test. The color change of the pH indicator was progressively suppressed by higher concentrations of serum albumin. CONCLUSIONS: Blood adversely affects the performance of the BUT. This is mediated by the buffering effect of serum albumin on the pH indicator, rather than by a direct inhibition on the urease activity.

Biopsy↗
PubMed1995

Growth inhibition of Ureaplasma urealyticum by the proton pump inhibitor lansoprazole: direct attribution to inhibition by lansoprazole of urease activity and urea-induced ATP synthesis in U. urealyticum.

The proton pump inhibitors (PPIs) omeprazole and lansoprazole and the acid-activated analog of lansoprazole AG-2000, which potently inhibit the urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), also inhibited the urease activities of cell-free extracts as well as intact cells of Ureaplasma urealyticum. The 50% inhibitory concentrations were between 1 and 25 microM. These compounds also inhibited the ATP synthesis induced by urea in ureaplasma cells. The 50% inhibitory concentrations for ATP synthesis were close to those for urease activity, but they were lower than those of urease inhibitors, such as acetohydroxamic acid, hydroxyurea, and thiourea. In addition, one of the metabolites of lansoprazole found in human urine, M-VI, also inhibited ureaplasmal urease activity and the ATP synthesis induced by urea at almost the same concentrations as those of lansoprazole. The inhibition of PPIs against ureaplasma urease was very similar to those against H. pylori urease, suggesting that the inhibitory mechanism against these ureases was due to the blockage of the SH residues on the cysteine of the enzyme. Omeprazole, lansoprazole, AG-2000, and M-VI inhibited the growth of U. urealyticum. Since ureaplasma urease is thought to be involved in the pathogenicity of this organism in the urogenital tract, PPIs and their analogs may be useful as chemotherapeutic agents against diseases caused by U. urealyticum.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1991

Proteus mirabilis urease: use of a ureA-lacZ fusion demonstrates that induction is highly specific for urea.

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated urinary tract infection, is the most frequent cause of infection-induced bladder and kidney stones. Urease-catalyzed urea hydrolysis initiates stone formation in urine and can be inhibited by acetohydroxamic acid and other structural analogs of urea. Since P. mirabilis urease is inducible with urea, there has been some concern that urease inhibitors actually induce urease during an active infection, thus compounding the problem of elevated enzyme activity. Quantitating induction by compounds that simultaneously inhibit urease activity has been difficult. Therefore, to study these problems, we constructed a fusion of ureA (a urease subunit gene) and lacZ (the beta-galactosidase gene) within plasmid pMID1010, which encodes an inducible urease of P. mirabilis expressed in E. coli JM103 (Lac-). The fusion protein, predicted to be 117 kDa, was induced by urea and detected on Western blots (immunoblots) with anti-beta-galactosidase antiserum. Peak beta-galactosidase activity of 9.9 mumol of ONPG (o-nitrophenyl-beta-D-galactopyranoside) hydrolyzed per min per mg of protein, quantitated spectrophotometrically, was induced at 200 mM urea. The uninduced rate was 0.2 mumol of ONPG hydrolyzed per min per mg of protein. Induction was specific for urea, as no structural analog of urea (including acetohydroxamic acid, hydroxyurea, thiourea, hippuric acid, flurofamide, or hydroxylamine) induced fusion protein activity. These data suggest that induction by inactivation of UreR, the urease repressor protein that governs regulation of the urease operon, is specific for urea and does not respond to closely related structural analogs.

Cloning, Molecular↗
PubMed1992

Characterization of Helicobacter pylori urease mutants.

The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs.

Bacterial Adhesion↗
PubMed1992

Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB.

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.

Apoenzymes↗
PubMed1993

Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli.

The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.

Amino Acids↗
PubMed1997

Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies.

Helicobacter pylori is a spiral, gram-negative bacterium which causes chronic gastritis and plays a critical role in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori expresses significant urease activity which is an essential virulence factor. Since a significant fraction of urease activity is located on the surface of the bacterium, the urease molecule is a logical choice as an antigen for a vaccine; currently recombinant urease apoenzyme is being tested as a vaccine in phase II clinical trials. We have recently demonstrated that urease and HspB (a homolog of the GroEL heat shock protein) become associated with the surface of H. pylori in vitro in a novel manner: these cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria, reflecting the unique characteristics of the outer membrane. To determine if similar mechanisms are operative in vivo, we determined the ultrastructural locations of urease and HspB within bacteria present in human gastric biopsies. Our results demonstrate that both urease and HspB are located within the cytoplasm of all bacteria examined in human gastric biopsies. Interestingly, a significant proportion of the bacteria examined also possessed variable amounts of surface-associated urease and HspB antigen (from 5 to 50% of the total antigenic material), indicating that in vivo, H. pylori has surface characteristics which enable it to adsorb cytoplasmic proteins. This is consistent with our altruistic autolysis model in which H. pylori uses genetically programmed bacterial autolysis to release urease and other cytoplasmic proteins which are subsequently adsorbed onto the surface of neighboring viable bacteria. These observations have important implications regarding pathogenesis and development of vaccines for H. pylori.

Bacterial Proteins↗
PubMed2006

The Helicobacter pylori urease B subunit binds to CD74 on gastric epithelial cells and induces NF-kappaB activation and interleukin-8 production.

The pathogenesis associated with Helicobacter pylori infection is the result of both bacterial factors and the host response. We have previously shown that H. pylori binds to CD74 on gastric epithelial cells. In this study, we sought to identify the bacterial protein responsible for this interaction. H. pylori urease from a pool of bacterial surface proteins was found to coprecipitate with CD74. To determine how urease binds to CD74, we used recombinant urease A and B subunits. Recombinant urease B was found to bind directly to CD74 in immunoprecipitation and flow cytometry studies. By utilizing both recombinant urease subunits and urease B knockout bacteria, the urease B-CD74 interaction was shown to induce NF-kappaB activation and interleukin-8 (IL-8) production. This response was decreased by blocking CD74 with monoclonal antibodies. Further confirmation of the interaction of urease B with CD74 was obtained using a fibroblast cell line transfected with CD74 that also responded with NF-kappaB activation and IL-8 production. The binding of the H. pylori urease B subunit to CD74 expressed on gastric epithelial cells presents a novel insight into a previously unrecognized H. pylori interaction that may contribute to the proinflammatory immune response seen during infection.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1988

Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.

Autoradiography↗
PubMed1991

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

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. pylori 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. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli 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.

Amino Acid Sequence↗
PubMed1992

Insertional inactivation of an Escherichia coli urease gene by IS3411.

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.

Base Sequence↗