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PubMed2005

Preparation and characterization of kappa-carrageenan immobilized urease.

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.

Carrageenan↗
PubMed2001

Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells.

The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.

Bacterial Adhesion↗
PubMed2003

Characterization and expression of a novel member (JBURE-II) of the urease gene family from jackbean [Canavalia ensiformis (L.) DC].

Canavalia ensiformis (jackbean) seeds contain the proteins urease and canatoxin, a variant form of the jackbean urease. Here we have cloned a cDNA encoding another isoform of urease, called JBURE-II. This cDNA was obtained by RT-PCR using as template total RNA extracted from C. ensiformis tissues. Nucleotide sequence analysis showed that JBURE-II clones share 86% similarity with known jackbean urease. The presence in C. ensiformis of a family of urease-related genes with at least three members was demonstrated by Southern blot analysis. In order to understand the pattern of expression of the JBURE-II gene, we collected tissue samples from different stages of flower and embryo development. The results of RT-PCR show that JBURE-II is expressed from flower buds throughout seed maturation. Semi-quantitative RT-PCR indicates that expression of urease and JBURE-II genes is induced in seedlings and in leaves treated with abscisic acid, a phytohormone involved in seed maturation and wound response. This work constitutes the first report on the presence of a family of urease genes in jackbean, and provides characterization of a cDNA encoding a new member of this gene family.

Abscisic Acid↗
PubMed1993

Isolation and characterization of urease from Aspergillus niger.

Urease was purified (4126-fold) from Aspergillus niger (NRRL 003) to a homologous enzyme preparation with a specific activity of 1341 mumol min-1 (mg protein)-1. One species of urease was detected in A. niger, with Km = 3.0 mM, native molecular mass 250,000 Da, pH optimum of 8.0 and a high specificity for urea. Hydroxyurea was a strong competitive inhibitor of urease activity, while N-methylurea acted as a weak uncompetitive inhibitor, based on Lineweaver-Burk and Eadie-Hoftstee plots. The activity of urease was enhanced by, but not dependent on, the presence of Na2EDTA, DL-dithiothreitol (< or = 0.1 to 5.0 mM), Ca2+, Ba2+ and citrate (2 to 20 mM). Urease activity was not affected by Na+, K+, Cl-, Br-, acetate or nitrate (2 to 20 mM), but was significantly decreased in the presence of Li+, Ni2+, Mg2+, Zn2+ or I-. Urease activity decreased 26.0% after 30 min at 65 degrees C, and 86.5% and 100.0% after 5 and 1 min at 80 and 100 degrees C, respectively. Urease activity decreased 30.5% after 90 d at 4 degrees C and 21.0% after 28 d at -20 or -80 degrees C.

Anions↗
PubMed2003

Medium pH-dependent redistribution of the urease of Helicobacter pylori.

Helicobacter pylori is an aetiological agent of gastric disease. Although the role of urease in gastric colonization of H. pylori has been shown, it remains unclear as to where urease is located in this bacterial cell. The purpose of this study was to define the urease-associated apparatus in the H. pylori cytoplasm. H. pylori was incubated at both a neutral and an acidic pH in the presence or absence of urea and examined by double indirect immunoelectron microscopy. The density of gold particles for UreA was greatest in the inner portion of the wild-type H. pylori cytoplasm at neutral pH but was greatest in the outer portion at acidic pH. This difference was independent of the presence of urea and was not observed in the ureI-deletion mutant. Also, the eccentric shift of urease in acidic pH was not observed in UreI. After a 2 day incubation period at acidic pH, it was observed that the urease gold particles in H. pylori assembled and were associated with UreI gold particles. Urease immunoreactivity shifted from the inner to the outer portion of H. pylori as a result of an extracellular decrease in pH. This shift was urea-independent and UreI-dependent, suggesting an additional role of UreI in urease-dependent acid resistance. This is the first report of the intracellular transport of molecules in bacteria in response to changes in the extracellular environment.

Bacterial Proteins↗
PubMed1992

Review article: urease, gastric ammonium/ammonia, and Helicobacter pylori--the past, the present, and recommendations for future research.

The presence of ammonium in gastric contents was described in 1852; urease activity in the stomach was identified 70 years later. The discovery of gastric urease resulted in intense research activity to discover its origin, function, and relation to the gastric levels of ammonium and urea. Interest in urease waned in the 1960s as most pertinent questions appeared to have been addressed and there was strong evidence that gastric urease was not a property of the stomach but was of microbial origin. Identification of Helicobacter pylori as the source of urease in the stomach in the last decade has resulted in a rebirth of interest in gastric urease and its products. There is little actual evidence to support a role for toxicity of ammonia in relation to H. pylori and the bulk of the evidence suggests that the products of urease activity are not toxic and may even be beneficial. The purpose of this review is to examine the older literature and to examine new findings in the perspective of what is already known and to suggest areas remaining to be examined. We ask, 'What is old, what is new, and what needs to be done?'

Ammonia↗
PubMed1993

Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes.

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.

Amino Acid Sequence↗
PubMed1993

Cloning, expression and sequencing of Helicobacter felis urease genes.

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.

Amino Acid Sequence↗
PubMed2005

Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque.

BACKGROUND/AIMS: Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. METHODS: The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. RESULTS: We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. CONCLUSION: Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.

Actinomyces↗
PubMed2006

gammadelta T cells increase with gastric mucosal interleukin (IL)-7, IL-1beta, and Helicobacter pylori urease specific immunoglobulin levels via CCR2 upregulation in Helicobacter pylori gastritis.

BACKGROUND AND AIMS: The purpose of this study was to investigate possible factors that could impact on gammadelta T cell accumulation in the gastric mucosa. METHOD: Subjects were 22 Helicobacter pylori (H. pylori)-free and 75 H. pylori-infected mucosa biopsies classified into grades I approximately III gastritis as per our previous study. The number of gammadelta- and 45 RO-positive T cells were determined by immunostaining. Gastric mucosal anti-H. pylori urease specific antibodies and interleukin (IL)-1beta, IL-2, 4, 7, 10 and IL-12 levels were assayed by enzyme-linked immunosorbent assay (ELISA). CC chemokine receptor 2 (CCR2) expression levels, migration, and cytokine production in gammadelta T cells stimulated by H. pylori urease were also evaluated. RESULTS: The gammadelta T cell count was significantly higher in grade III gastritis which exhibits strong immunoglobulin (Ig)A and IgG responses to H. pylori urease with lymphoid follicles than in other groups. gammadelta T cell count was significantly correlated with IL-1beta and interleukin-7 (IL-7) levels in the gastric mucosa. H. pylori urease immunoreactivity was detected in lamina propria of grade III gastritis, along with many gammadelta T cells. After H. pylori eradication therapy, the gammadelta T cell count in grade III gastritis significantly decreased. H. pylori urease stimulated significant increases in CCR2 expression levels, although to a lesser degree than those induced by IL-7 stimulation in both peripheral and mucosal gammadelta T cells. Interferon (IFN)-gamma and IL-10 production was also stimulated by H. pylori urease in peripheral gammadelta T cells. CONCLUSIONS: Gastric mucosal increases in IL-7 and IL-1beta closely corresponded to the accumulation of gammadelta T cells in gastric mucosa. An association was also seen between gammadelta T cell accumulation and H. pylori urease-specific Ig levels.

Adult↗
PubMed1997

A novel mechanism of urease regulation in Yersinia enterocolitica.

Yersinia enterocolitica produces the enzyme urease which hydrolyses urea, resulting in the production of carbonic acid and ammonia and a net increase in pH. In the presence of urea, urease enhances survival of Y. enterocolitica in the stomach and presumably in other acidic environments the bacteria encounter during the course of infection. In this study we show that Y. enterocolitica urease is a cytosolic enzyme which has a low Km value (0.15 +/- 0.01 mM urea), suggesting that it functions at close to maximum velocity even at the low concentrations of urea available to Y. enterocolitica in gastric fluid and other tissues. Y. enterocolitica urease was active over a wide pH range, but unlike most other bacterial ureases, displayed an optimal activity at pH 3.5-4.5, suggesting a physiological role in protecting the bacteria from acid. Higher levels of urease activity were attained at 28 degrees C than at 37 degrees C, and investigation of the regulation of urease production revealed that the enzyme was not induced by urea, or by nitrogen limitation. Instead maximal activity was attained during the stationary phase of growth which coincides with the period of maximum acid tolerance of the bacteria. This type of regulation has not been described for any other ureolytic bacteria and seems to be unique to Y. enterocolitica.

Bacterial Proteins↗
PubMed2002

Transcriptional and mutational analysis of the Helicobacter pylori urease promoter.

Urease is an essential virulence factor of the human gastric pathogen Helicobacter pylori, and is expressed to very high levels. The promoter of the urease operon contains sequences resembling the canonical -10 and extended -10 motifs, but no discernible -35 motif. To establish the role of different motifs and regions in the urease promoter, we fused the urease promoter to a genomic lacZ reporter gene in H. pylori, made substitutions in the aforementioned promoter motifs, and also made deletions in the upstream sequences removing regulatory sequences. Substitutions in the -10, extended -10 and predicted -35 motifs all significantly altered expression of the lacZ reporter gene, demonstrating their importance in transcription of the H. pylori urease operon. In contrast, sequential deletions upstream of the -35 region did not affect expression of the lacZ reporter gene. This demonstrates the modular structure of the H. pylori urease promoter, where basal levels of transcription are initiated from a typical sigma(70) promoter, which requires -10 and extended -10 motifs, and also its -35 motif for efficient transcription. Upstream sequences are not involved in basal levels of urease transcription, but play an important role in responses to environmental stimuli like nickel.

Amino Acid Motifs↗
PubMed1997

[Urease-negative Helicobacter pylori isolates from gastrointestinal mucosa of patients with peptic ulcer].

It is well known that Helicobacter pylori produced a large amount of urease which plays an important role in the maintenance of infection and adhesion. Two thousand three hundred and thirty-one specimens of the gastrointestinal mucosa were obtained from patients with peptic ulcer to isolate H. pylori strains. Of these specimens, 1602 strains of H. pylori were isolated and 7 urease-negative H. pylori strains were found. Biological characteristics (except urease production) of urease-negative H. pylori were in accord with urease-positive reference strains (ATCC strains). Furthermore, these urease-negative strains were confirmed to be H. pylori strains by PCR. These results suggest that the emergence of urease-negative strains pose a new problem for H. pylori infection in patients with peptic ulcer.

Gastric Mucosa↗
PubMed1995

Requirement of carbon dioxide for in vitro assembly of the urease nickel metallocenter.

Assembly of protein metallocenters is not well understood. Urease offers a tractable system for examination of this process. Formation of the urease metallocenter in vivo is known to require four accessory proteins: UreD, postulated to be a urease-specific molecular chaperone; UreE, a nickel(II)-binding protein; and UreF and UreG, of unknown function. Activation of purified Klebsiella aerogenes urease apoprotein was accomplished in vitro by providing carbon dioxide (half-maximal activation at approximately 0.2 percent carbon dioxide) in addition to nickel ion. Activation coincided with carbon dioxide incorporation into urease in a pH-dependent reaction (pKa > or = 9, where Ka is the acid constant). The concentration of carbon dioxide also affected the amount of activation of UreD-urease apoprotein complexes. These results suggest that carbon dioxide binding to urease apoprotein generates a ligand that facilitates productive nickel binding.

Apoproteins↗
PubMed1981

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.

Ammonium Chloride↗
PubMed1987

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Cryptococcus↗
PubMed1993

Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a significant cause of bacteriuria and acute pyelonephritis in humans, produces urease. This high-molecular-weight, multimeric, cytoplasmic enzyme hydrolyzes urea to ammonia and carbon dioxide. To assess the role of urease in colonization, urolithiasis, and acute pyelonephritis in an animal model of ascending urinary tract infection, we compared a uropathogenic strain of P. mirabilis with its isogenic urease-negative mutant, containing an insertion mutation within ureC, the gene encoding the large subunit of the enzyme. Mice challenged transurethrally with the parent strain developed significant bacteriuria and urinary stones. The urease-negative mutant had a 50% infective dose of 2.7 x 10(9) CFU, a value more than 1,000-fold greater than that of the parent strain (2.2 x 10(6) CFU). The urease-positive parent strain reached significantly higher concentrations and persisted significantly longer in the bladder and kidney than did the mutant. Indeed, in the kidney, the parent strain increased in concentration while the mutant concentration fell so that, by 1 week, the parent strain concentration was 10(6) times that of the mutant. Similarly, the urease-positive parent produced significantly more severe renal pathology than the mutant. The initial abnormalities were in and around the pelvis and consisted of acute inflammation and epithelial necrosis. By 1 week, pyelitis was more severe, crystals were seen in the pelvis, and acute pyelonephritis, with acute interstitial inflammation, tubular epithelial cell necrosis, and in some cases abscesses, had developed. By 2 weeks, more animals had renal abscesses and radial bands of fibrosis. We conclude that the urease of P. mirabilis is a critical virulence determinant for colonization, urolithiasis, and severe acute pyelonephritis.

Acute Disease↗
PubMed1996

Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus.

The hydrolysis of urea by urease enzyme of oral bacteria is believed to have a major impact on oral microbial ecology and to be intimately involved in oral health and diseases. To begin to understand the biochemistry and genetics of oral ureolysis, a study of the urease of Streptococcus salivarius, a highly ureolytic organism which is present in large numbers on the soft tissues of the oral cavity, has been initiated. By using as a probe a 0.6-kpb internal fragment of the S. salivarius 57.I ureC gene, two clones from subgenomic libraries of S. salivarius 57.I in an Escherichia coli plasmid vector were identified. Nucleotide sequence analysis revealed the presence of one partial and six complete open reading frames which were most homologous to ureIAB-CEFGD of other ureolytic bacteria. Plasmid clones were generated to construct a complete gene cluster and used to transform E. coli and Streptococcus gordonii DL1, a nonureolytic, dental plaque microorganism. The recombinant organisms expressed high levels of urease activity when the growth medium was supplemented with NiCl2. The urease enzyme was purified from E. coli, and its biochemical properties were compared with those of the urease produced by S. salivarius and those of the urease produced by S. gordonii carrying the plasmid-borne ure genes. In all cases, the enzyme had a Km of 3.5 to 4.1 mM, a pH optimum near 7.0, and a temperature optimum near 60 degrees C. S. gordonii carrying the urease genes was then demonstrated to have a significant capacity to temper glycolytic acidification in vitro in the presence of concentrations of urea commonly found in the oral cavity. The ability to genetically engineer plaque bacteria that can modulate environmental pH through ureolysis will open the way to using recombinant ureolytic organisms to test hypotheses regarding the role of oral ureolysis in dental caries, calculus formation, and periodontal diseases. Such recombinant organisms may eventually prove useful for controlling dental caries by replacement therapy.

Base Sequence↗