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PubMed1992

Structural comparison of urease and a GroEL analog from Helicobacter pylori.

Electron microscopy of purified protein preparations indicated that Helicobacter pylori urease consisted of circular particles that are 13 nm in diameter, some of which showed indications of threefold rotational symmetry. A GroEL analog of H. pylori (Hp60K) appeared as a disc-shaped molecule with a diameter similar to that of urease but possessed sevenfold rotational symmetry. In a side-view projection, Hp60K appeared as two or four discs stacked side by side.

Amino Acid Sequence↗
PubMed2000

Dual functions of Streptococcus salivarius urease.

A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at the ureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivarius could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.

Gene Deletion↗
PubMed2001

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity.

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

ATP-Binding Cassette Transporters↗
PubMed2001

Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology.

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

Antibodies, Bacterial↗
PubMed2005

Purification and properties of the Klebsiella aerogenes UreE metal-binding domain, a functional metallochaperone of urease.

Klebsiella aerogenes UreE, a metallochaperone that delivers nickel ions during urease activation, consists of distinct "peptide-binding" and "metal-binding" domains and a His-rich C terminus. Deletion analyses revealed that the metal-binding domain alone is sufficient to facilitate urease activation. This domain was purified and shown to exhibit metal-binding properties similar to those of UreE lacking only the His-rich tail.

Bacterial Proteins↗
PubMed1962

Urease activity of Proteus species in filtered compared with autoclaved urea media.

Hatch, M. H. (Brown University, Providence, R.I.) and C. A. Stuart. Urease activity of Proteus species in filtered compared with autoclaved urea media. J. Bacteriol. 83:1119-1123. 1962.-Urease activity of a number of strains of four Proteus species was compared in a filtered medium and an autoclaved medium. Various amounts of saline suspensions of the organisms were used as inocula for the two media. In some experiments Seitz-filtered yeast extract was added to autoclaved basal urea medium. All tests were incubated at 35 C and read 8, 12, 24, and 48 hr after inoculation. The filtered medium appeared superior to the autoclaved medium, particularly when small inocula were employed. Under these conditions, many tests with all four species were negative in the autoclaved medium at 48 hr, in contrast to 100% positive reactions in the filtered medium with all species except P. morganii. The experiments in which Seitz-filtered yeast extract was added to autoclaved basal urea medium suggested that both destruction of growth-promoting substances in the yeast extract and production of conditions inhibitory for growth during autoclaving might be involved in the differences observed between the two media.

Culture Media↗
PubMed1975

Use of urease test disks in the identification of mycobacteria.

Use of commercially available urea differentiation disks is a rapid and convenient means of determining the urease activity of mycobacteria. In the study performed, paper disks containing Ewing's Urea R Broth were compared with two other methods of testing for urease production. Over 1,500 tests were performed with recent patient isolates from which at least 15 different species of mycobacteria were identified. The tests were read at intervals for 72 h, and a comparison of test results as to rapidity and accuracy for each species was tabulated. The urea disks showed a faster reaction time when compared with the other two methods. They also showed a high percentage (79 to 100%) of reliability for each species on the first run, based on the expected response.

Classification↗
PubMed1984

Stone formation by Ureaplasma urealyticum in human urine and its prevention by urease inhibitors.

When Ureaplasma urealyticum T-960 was inoculated into normal human urine (10(8) viable cells per ml of urine), a white precipitate formed, with an increase in pH of the infected urine. This precipitate was identified as a mixture of struvite and whitelockite by analysis of the infrared spectrum. Its formation was completely prevented by the addition of 10 microM N-benzoylphosphotriamide, 20 microM N-isopentenoylphosphotriamide, or 0.5 mM caprylohydroxamic acid without the alkalinization of the urine, and the Ureaplasma color change units were also decreased markedly by these compounds. The apparent concentrations for 50% inhibition by N-benzoylphosphotriamide,N-isopentenolyphosphotriamide, and caprylohydroxamic acid against Ureaplasma urease were 7 nM, 2 nM, and 2.2 microM, respectively. From these results, it seems that stone formation by U. urealyticum is prevented with these compounds, that prevention being directly attributable to the inhibition of urease activity, which causes the death of the cells.

Bacteriuria↗
PubMed1986

o-Nitrophenyl-beta-D-galactopyranoside-urease-indole broth, a new composite tube medium for Salmonella screening.

A new composite broth medium combining o-nitrophenyl-beta-D-galactopyranoside (ONPG) and urease and indole tests in a single tube is described. High-level agreement with individual conventional tests was recorded in comparative studies with 2,412 cultures of members of the family Enterobacteriaceae, i.e., 100% agreement with the exception of Hafnia spp. (96.3% agreement) for the ONPG test and Citrobacter, Enterobacter, and Hafnia spp. (75, 86.4, and 98.2% agreement, respectively) for the urease test. The new medium seems especially promising as a screen for Salmonella subgroup I which encompasses most pathogenic Salmonella species other than the Arizona subgroup.

Culture Media↗
PubMed1988

Urease-positive thermophilic Campylobacter (Campylobacter laridis variant) isolated from an appendix and from human feces.

Urease-positive thermophilic campylobacters were isolated for the first time from the feces of two adults with diarrheal disease and from the appendix of a child with appendicitis. They were identified as Campylobacter laridis by a hybridization dot blot assay. Urease testing should be included in the tests used for the identification of campylobacters at the species level, even for those strains which are not of gastric origin.

Appendix↗
PubMed1992

Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates.

Helicobacter pylori has been demonstrated as an etiologic agent of human gastritis and peptic ulcer formation. However, there is no straightforward basis to distinguish different isolates. We used the polymerase chain reaction (PCR) to amplify the urease structural subunit genes, ureA and ureB, which, when digested with appropriate restriction endonucleases, allow the differentiation of patterns on agarose gels. PCR amplification was possible with DNA rapidly extracted from H. pylori by alkaline lysis and phenol-chloroform. The 2.4-kb PCR products amplified from 22 clinical isolates and subjected to HaeII restriction endonuclease digestion produced 10 distinct patterns on agarose gels, with two patterns being shared between five and six strains. PCR amplification of the urease genes may enable the differentiation of closely related H. pylori strains by restriction digest analysis of PCR-amplified ureA and ureB genes.

Bacterial Typing Techniques↗
PubMed1997

Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the West Coast of the United States.

Urease-positive (Ure+) and urease-negative (Ure-) strains of Vibrio parahaemolyticus isolated from patients on the West Coast of the United States between 1979 and 1995 were analyzed for the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes (trh1 and trh2). The DNA colony hybridization method with the polynucleotide probes was used to determine the distribution of the genes. Of 60 Ure+ strains, 59 strains (98%) had the trh (either trh1 or trh2) gene and 54 strains (90%) carried the tdh gene. The absence of the trh gene or a related sequence in an exceptional Ure+ strain was confirmed by Southern blot analyses. The stronger correlation with the trh gene than with the tdh gene was mostly attributable to strains possessing only the trh2 gene. Of 25 Ure- strains, 20 strains (80%) had the tdh gene but none had the trh gene. These results indicate a very strong correlation between the Ure+ phenotype and the trh gene and are consistent with those reported for strains isolated in Asia. The Ure+ strains carrying the trh genes were not restricted to a unique group of the strains. The O4:K12 strains carrying the trh1 gene have predominantly been isolated since 1979. However, strains of various non-O4:K12 serovars carrying either the trh1 or the trh2 gene became predominant after 1992. In addition, analysis by the arbitrarily primed PCR method revealed two subgroups within the selected Ure+ O4:K12 strains. Hybridization tests with oligonucleotide probes demonstrated that the trh1 sequences of the West Coast strains differ to some extent from those of Asian strains. Nevertheless, a PCR method previously established to detect both the trh1 and the trh2 genes in Asian strains could detect 98% of those genes in the West Coast strains.

Bacterial Proteins↗
PubMed2001

Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture.

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only approximately 10(3) CFU/ml. All H. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), beta-cyclodextrin (200 microg/ml), or cholesterol (50 microg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 microg/ml), beta-cyclodextrin (200 microg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or beta-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.

Animals↗
PubMed2001

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species.

Over the past decade, several Helicobacter species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents. In this report, we describe a novel urease-negative helicobacter, Helicobacter typhlonius sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections. H. typhlonius is genetically most closely related to H. hepaticus. Like H. hepaticus, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H. hepaticus. Notably, H. typhlonius does not produce urease or H(2)S nor does it hydrolize indoxyl-acetate. Compared to other Helicobacter species that commonly colonize rodents, H. typhlonius was found to be less prevalent than H. hepaticus and H. rodentium but as prevalent as H. bilis. H. typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

Animals↗
PubMed2001

Association of the urease gene with enterohemorrhagic Escherichia coli strains irrespective of their serogroups.

Among various diarrheagenic Escherichia coli strains from clinical sources, we found that the urease gene was specifically associated with enterohemorrhagic E. coli (EHEC) strains irrespective of their serogroups. The results suggest that the urease gene can be a useful genetic marker for the detection of EHEC strains and for the diagnosis of infections caused by EHEC strains in the clinical situation.

Diarrhea↗
PubMed1993

Development of a 14C-urea breath test in ferrets colonised with Helicobacter mustelae: effects of treatment with bismuth, antibiotics, and urease inhibitors.

A 14C-urea breath test analogous to its clinical counterpart is described for use in ferrets naturally or experimentally infected with Helicobacter mustelae. The test is performed within a sealed glass metabolism chamber through which air is drawn at a constant rate and expired breath collected into sodium hydroxide. Peak 14CO2 production occurred approximately 1 hour after substrate administration. Both inter- and intra-animal responses were highly reproducible, with mean coefficients of variation less than 10%. Other than enhancing peak 14CO2 levels very slightly, fasting had little influence on the response. In infant animals challenged with H mustelae, breath test activity increased linearly with the total count of culturable bacteria isolated from the antrum. Treatment of established infections with colloidal bismuth subcitrate (DeNol) for 4 weeks resulted in clearance of all detectable bacteria but retention of some breath test activity. Subsequent regrowth of bacteria was parallelled by an increase in the breath test response. Inclusion of amoxycillin and metronidazole in the treatment regimen, however, eradicated all the bacteria and almost totally eliminated 14CO2 production. This response parallels the clinically observed suppressive effect on H pylori achieved with bismuth alone relative to the total eradication seen with triple therapy. A single oral dose of the urease inhibitor, flurofamide, inhibited over 90% of the response for at least 24 hours. Acetohydroxamic acid was less effective. These findings suggest that in the ferret H mustelae model, breath test analysis can be a useful, non-invasive alternative to endoscopy for evaluation of agents affecting either growth of the organism or urease activity.

Animals↗
PubMed1991

Harnessing of urease activity of Helicobacter pylori to induce self-destruction of the bacterium.

Eradication of Helicobacter pylori with currently available antibacterial agents is unsatisfactory due to the risk of side-effects and the emergence of resistance. The organism rapidly dies in vitro in the presence of urea at pH 6. When incubated in citrate buffer (pH 6) plus urea (10 mM) the five minute survival was 26% compared with 96% without urea and the survival progressively decreased with increasing urea concentrations, being only 9% in 50 mM urea. The bactericidal effect depended on pH as the organism survived in citrate buffer (pH 7) plus urea (50 mM). The death of the organism at pH 6 in the presence of urea was prevented by the addition of the competitive urease inhibitor hydroxyurea. These findings indicate that destruction of the organism is mediated by its exceptionally high urease activity. Harnessing this enzyme to induce self-destruction could provide a new approach to eradicating this common infection.

Buffers↗