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The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC 50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC 50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol -1 ; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.

Understanding the functional potential of the gut microbiome is of primary importance for the design of innovative strategies for allergy treatment and prevention. Here we report the gut microbiome features of 90 children affected by food (FA) or respiratory (RA) allergies and 30 age-matched, healthy controls (CT). We identify specific microbial signatures in the gut microbiome of allergic children, such as higher abundance of Ruminococcus gnavus and Faecalibacterium prausnitzii, and a depletion of Bifidobacterium longum, Bacteroides dorei, B. vulgatus and fiber-degrading taxa. The metagenome of allergic children shows a pro-inflammatory potential, with an enrichment of genes involved in the production of bacterial lipo-polysaccharides and urease. We demonstrate that specific gut microbiome signatures at baseline can be predictable of immune tolerance acquisition. Finally, a strain-level selection occurring in the gut microbiome of allergic subjects is identified. R. gnavus strains enriched in FA and RA showed lower ability to degrade fiber, and genes involved in the production of a pro-inflammatory polysaccharide. We demonstrate that a gut microbiome dysbiosis occurs in allergic children, with R. gnavus emerging as a main player in pediatric allergy. These findings may open new strategies in the development of innovative preventive and therapeutic approaches. Trial: NCT04750980., (© 2021. The Author(s).)

Yvh1 is a dual-specificity phosphatase (DUSP) that is evolutionarily conserved in eukaryotes, including yeasts and humans. Yvh1 is involved in the vegetative growth, differentiation, and virulence of animal and plant fungal pathogens. All Yvh1 orthologs have a conserved DUSP catalytic domain at the N-terminus and a zinc-binding (ZB) domain with two zinc fingers (ZFs) at the C-terminus. Although the DUSP domain is implicated in the regulation of MAPK signaling in humans, only the ZB domain is essential for most cellular functions of Yvh1 in fungi. This study aimed to analyze the functions of the DUSP and ZB domains of Yvh1 in the human fungal pathogen Cryptococcus neoformans, whose Yvh1 (CnYvh1) contains a DUSP domain at the C-terminus and a ZB domain at the N-terminus. Notably, CnYvh1 has an extended internal domain between the two ZF motifs in the ZB domain. To elucidate the function of each domain, we constructed individual domain deletions and swapping strains by complementing the yvh1Δ mutant with wild-type (WT) or mutated YVH1 alleles and examined their Yvh1-dependent phenotypes, including growth under varying stress conditions, mating, and virulence factor production. Here, we found that the complementation of the yvh1Δ mutant with the mutated YVH1 alleles having two ZFs of the ZB domain, but not the DUSP and extended internal domains, restored the WT phenotypic traits in the yvh1Δ mutant. In conclusion, the ZB domain, but not the N-terminal DUSP domain, plays a pivotal role in the pathobiological functions of cryptococcal Yvh1.

Dental diseases are among the common health issues experienced around the world. Dental caries is one of the most predominant oral diseases worldwide. Major factors associated with caries development include poor oral hygiene, the content of specific carbohydrates in the diet, dental biofilm formation, the cariogenic microbial load, reduction in salivary flow, insufficient fluoride exposure, gingival recession, genetic factors, and lack of personal attention to one's dental health. Several preventive measures have been implemented to reduce the risk of the development of caries. Probiotics are live microbes that when administered in suitable amounts confer health benefits on the host; they are recognized as potential adjunct therapeutic agents for several diseases. The present manuscript summarizes recent findings on the role of probiotics in dental caries prevention and the possible mechanisms of probiotic effects. Review of the literature indicates the regular consumption of probiotic products significantly reduced the risk of caries by inhibiting cariogenic bacteria and enriching commensal microbes in the oral cavity. Buffering the salivary pH, production of bacteriocin and enzymes (dextranase, mutanase, and urease), the capacity of competing for the adhesion and colonization on tooth surfaces are the possible mechanisms behind the beneficial effect of probiotics. Further studies are necessary to address the efficacy of long-term probiotic supplementation on the control of dental diseases and the influence of childhood probiotic supplementation on the risk of caries development.

Species of fungi belonging to the order Mucorales can be found everywhere in the environment. Gilbertella persicaria, which belongs to this order, have often been isolated from fruits and in water systems. However, there has been no report of isolation of this fungus from human samples. During a gut mycobiome study, from the Segamat community, Gilbertella persicaria was isolated from a human fecal sample and was characterized through a series of morphological assessment, biochemical tests, and molecular techniques. The isolate produced a white velvety surface that turned grayish after 24 h. Although no biofilm production was observed, the results indicated that the isolate could form calcium oxalate crystals, produced urease, and was resistant to low pH. The isolate was sensitive to amphotericin but resistant to voriconazole and itraconazole. The features of this fungus that could help in its survival in the human gut are also discussed.

Recent research suggests that protein deficiency symptoms are influenced by the intestinal microbiota. We investigated the influence of low protein diet on composition of the intestinal microbiota through animal experiments. Specific pathogen-free (SPF) mice were fed one of four diets (3, 6, 9, or 12% protein) for 4 weeks (n = 5 per diet). Mice fed the 3% protein diet showed protein deficiency symptoms such as weight loss and low level of blood urea nitrogen concentration in their serum. The intestinal microbiota of mice in the 3% and 12% protein diet groups at day 0, 7, 14, 21 and 28 were investigated by 16S rRNA gene sequencing, which revealed differences in the microbiota. In the 3% protein diet group, a greater abundance of urease producing bacterial species was detected across the duration of the study. In the 12% diet protein group, increases of abundance of Streptococcaceae and Clostridiales families was detected. These results suggest that protein deficiency may be associated with shifts in intestinal microbiota.

In the present study, we investigated the role of calcite, i.e., microbiologically-induced precipitate by ureolytic Trichoderma sp. MG, in remediation of soils contaminated with arsenic (As) and lead (Pb). The fungus tolerates high concentrations of As (500 mg/L) and Pb (650 mg/L). The effects of three factors, i.e., urea concentration, CaCl 2 concentration and pH, on urease production and bio-mineralization of As and Pb were investigated using Box-Behnken design. The maximum urease production (920 U/mL) and metal removal efficiency (68% and 59% for Pb and AS, respectively) were observed in the medium containing urea of 300 mM and CaCl 2 of 75 mM at pH 9.0. Fourier transform infrared spectroscopy result revealed the formation of metal carbonates by the isolate MG. Sequential extraction of metals revealed that the carbonate fractions of As and Pb were increased to 46.4% and 42.4% in bioremediated soil, whereas in control they were 35.5% and 32.5%, respectively. The X-ray powder diffraction result further confirmed the role of calcite precipitate in bioremediation of As- and Pb-contaminated soils. The results points out that the microbiologically-induced calcite precipitation is a feasible, eco-friendly technology for the bioremediation of As- and Pb-contaminated sites., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rhodotorula yeasts are pink, encapsulated basidiomycetes isolated from a variety of environments and clinical settings. They are increasingly linked with disease, particularly central venous catheter infections and meningitis, in immunocompromised patients. Eight clinical and eight environmental strains molecularly typed as Rhodotorula mucilaginosa were compared to six Cryptococcus neoformans strains for phenotypic variability. Growth on cell integrity-challenging media suggested that R. mucilaginosa cells possess differences in signaling pathways, cell wall composition, or assembly and that their membranes are more susceptible to perturbations than those of C. neoformans All 16 R. mucilaginosa strains produced urease, while none produced melanin with l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. India ink staining reveals that clinical R. mucilaginosa capsules are larger than environmental capsules but that both are generally smaller than C. neoformans capsules. All R. mucilaginosa strains were resistant to fluconazole. Only two clinical strains were susceptible to voriconazole; all of the environmental strains were resistant. We generated an anticapsular antibody (Rh1) to R. mucilaginosa ; Rh1 did not bind C. neoformans control strains, was specific to Rhodotorula species, and bound to all tested Rhodotorula strains. Binding assays performed with wheat germ agglutinin (WGA), concanavalin A (ConA), calcofluor white (CFW), and eosin Y dye (EY) cell surface probes suggested that chitin may be more accessible in R. mucilaginosa but that the total abundance of chitooligomers is less than in C. neoformans This report describes a novel reagent that can be used to identify Rhodotorula species and lays the foundation for future cell envelope composition analysis. IMPORTANCE Currently, there is very little known about the phenotypic variability within species of Rhodotorula strains and the role of their capsule. Cryptococcus neoformans has been considered the only encapsulated human fungal pathogen, but as more individuals come to live in states of immunocompromised health, they are more susceptible to fungal infections, including those by Rhodotorula R. mucilaginosa species are some of those most commonly associated with clinical infections. We wanted to know if clinical and environmental strains of R. mucilaginosa demonstrated disparate capsule phenotypes. With limited antifungal options available and clinical Rhodotorula spp. often resistant to common antifungal drugs such as fluconazole, caspofungin (1, 2), and voriconazole (2), a better understanding of the fungal biology could inform the design and use of future antifungal drugs. The generation of an antibody specific to Rhodotorula fungi could be a useful diagnostic tool, and this work presents the first mention of such in the literature., (Copyright © 2019 Yockey et al.)

Cadmium (Cd) is a highly toxic metal that can affect human health and environmental safety. The purpose of this study was to research the removal of Cd from an environmental perspective. In this article, four highly urease-active strains (CZW-2, CZW-5, CZW-9 and CZW-12) were isolated from an abandoned mine and their phylogenetic trees were analyzed. The maximum enzyme activities, the mineralized precipitate and the removal rates of these strains were compared. The results showed that CZW-2 had the highest urease activity at 51.6 U/mL, and the removal rates of CZW-2, CZW-5, CZW-9 and CZW-12 after 120 h were 80.10%, 72.64%, 76.70% and 73.40%, with an initial concentration of Cd of 2 mM in the Cd precipitation experiments. XRD (X-ray diffractometer), EDS (Energy dispersive spectrometer) and FTIR (Fourier transform infrared spectroscopy) analysis indicated that the mineralized precipitate was CdCO₃. SEM (Scanning electron microscopy) analysis revealed that the diameter of the oval-shaped mineralized product ranked from 0.5 to 2 μm. These strains were used to remedy Cd-contaminated soil, and five different fractions of Cd were measured. Compared with the control, the results of spraying pre-cultured strains containing 2% urea to remove Cd from contaminated soils showed that the exchangeable fraction of Cd decreased by 53.30%, 27.78%, 42.54% and 53.80%, respectively, whereas the carbonate-bound fraction increased by 55.42%, 20.27%, 39.67% and 34.36%, respectively, after one month. These data show that these strains can effectively reduce the bioavailability and mobility of Cd in contaminated soils. The results indicate that biomineralization based on the decomposition of substrate urea can be applied to remedy heavy contaminated soil and water.

Helicobacter pylori is a human pathogen that infects the stomach, where it experiences variable pH. To survive the acidic gastric conditions, H. pylori produces large quantities of urease, a nickel enzyme that hydrolyzes urea to ammonia, which neutralizes the local environment. One of the regulators of urease expression in H. pylori is HpNikR, a nickel-responsive transcription factor. Here we show that HpNikR also regulates urease expression in response to changes in pH, linking acid adaptation and nickel homeostasis. Upon measuring the cytosolic pH of H. pylori exposed to an external pH of 2, similar to the acidic shock conditions that occur in the human stomach, a significant drop in internal pH was observed. This decrease in internal pH resulted in HpNikR-dependent activation of ureA transcription. Furthermore, analysis of a slate of H. pylori genes encoding other acid adaptation or nickel homeostasis components revealed HpNikR-dependent regulation in response to acid shock. This regulation was consistent with pH-dependent DNA binding to the corresponding promoter sequences observed in vitro with purified HpNikR. These results demonstrate that HpNikR can directly respond to changes in cytosolic pH during acid acclimation and illustrate the exquisitely coordinated regulatory networks that support H. pylori infections in the harsh environment of the human stomach., Competing Interests: The authors declare no conflict of interest.

In this study, the feasibility of introducing calcite-forming bacteria into concrete pavements to improve their mechanical performance was investigated. Lysinibacillus sphaericus WJ-8, which was isolated in a previous study and is capable of exhibiting high urease activity and calcite production, was used. When analyzed via scanning electron microscopy (SEM) and X-ray diffraction, WJ-8 showed a significant amount of calcite precipitation. The compressive strength of cement mortar mixed with WJ-8 cells and nutrient medium (urea with calcium lactate) increased by 10% compared with that of the controls. Energy dispersive x-ray spectroscopy analyses confirmed that the increase in strength was due to the calcite formed by the WJ-8 cells.

Proteus mirabilis is a model organism for urease-producing uropathogens. These diverse bacteria cause infection stones in the urinary tract and form crystalline biofilms on indwelling urinary catheters, frequently leading to polymicrobial infection. Recent work has elucidated how P. mirabilis causes all of these disease states. Particularly exciting is the discovery that this bacterium forms large clusters in the bladder lumen that are sites for stone formation. These clusters, and other steps of infection, require two virulence factors in particular: urease and MR/P fimbriae. Highlighting the importance of MR/P fimbriae is the cotranscribed regulator, MrpJ, which globally controls virulence. Overall, P. mirabilis exhibits an extraordinary lifestyle, and further probing will answer exciting basic microbiological and clinically relevant questions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A 79-year-old woman was admitted emergently for disturbance of consciousness. Her consciousness level was Japan coma scale 20, and she presented with hypermyotonia. Brain magnetic resonance imaging and cerebrospinal fluid examination showed normal findings. Her blood tests showed an increased ammonia level of 291 μg/dl with normal liver function. We catheterized the bladder for urinary retention. Eight hours after admission, the blood level of ammonia decreased to 57 μg/dl and the patient's consciousness level improved. Corynebacterium pseudodiphtheriticum, which is a bacteria producing urease, was detected from a urine culture. It is important to recognize that obstructive urinary tract infection caused by urease-producing bacteria can cause hyperammonemia.

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

A 71-year-old woman with a 9-year history of Parkinson's disease was admitted to our hospital emergently because of consciousness disturbance. Her consciousness level was 200 on the Japan coma scale (JCS), and she presented with tenderness and distension of the lower abdomen. Brain computed tomography showed normal findings. Blood tests showed an increased ammonia level (209 μg/dl) with normal AST and ALT levels. We catheterized the bladder for urinary retention. Five hours after admission, the blood ammonia level decreased to 38 μg/dl, and her consciousness level improved dramatically. Corynebacterium urearyticum, a bacterial species that produces urease, was detected by urine culture. Therefore, she was diagnosed with hyperammonemic encephalopathy resulting from urinary tract infection caused by urease-producing bacteria. In this case, urologic active agents had been administered to treat neurogenic bladder. We suspect that these drugs caused urinary obstruction and urinary tract infection. It is important to recognize that obstructive urinary tract infection caused by urease-producing bacteria can cause hyperammonemia. Neurological disorders, such as Parkinson's disease, tend to complicate neurogenic bladder. This disease should be considered in elderly patients with Parkinson's disease who are receiving urologic active drugs.

We present here a rare case of hyperammonemia without liver dysfunction or portal-systemic shunting. The patient was an 80-year-old woman with a history of neurogenic bladder. She was admitted to a nearby hospital for vomiting, diarrhea and consciousness disturbance. Two days after admission, she was transferred to our hospital because of persistant consciousness disturbance. Laboratory data revealed hyperammonemia, but there was no indication of liver dysfunction. Moreover abdominal computed tomography did not reveal any clear finding of liver disease or portal-systemic shunting, but we noted multiple large bladder diverticula. Antibiotic therapy, tracheal intubation, ventilator management and bladder catheterization were performed. The patient's level of consciousness improved rapidly. Urinary culture revealed Bacteroides ureolyticus (urease-producing bacteria). The patient was diagnosed with hyperammonemia and a urinary tract infection due to urease-producing bacteria. Thus, physicians should be aware that obstructive urinary tract infections due to urease-producing bacteria can also be the cause of hyperammonemia.

The antimicrobial effects of methanol and ethanol extracts of Inula britannica against several Helicobacter pylori strains (26695, J99, and SS1) were evaluated in vitro, to determine their applicability as functional foods. In the paper disc diffusion method, the antimicrobial effects of the I. britannica extracts against the H. pylori strains were apparent. Viable cell counting also showed that the extracts at 100 μg/ml concentration dramatically decreased the viability of the H. pylori strains. In particular, the methanol and ethanol extracts at a concentration of 100 μg/ml reduced the H. pylori SS1 cell number to 2.46 log CFU/ml and 1.08 log CFU/ml, respectively. In the presence of 100 μg/ml extracts, the urease production of H. pylori SS1 was decreased to more than 30%, whereas that of H. pylori J99 and H. pylori 26695 was decreased to about 20%, relative to the controls. The extracts inhibited the attachment of the H. pylori strains to human gastric AGS cells as well as caused the detachment of already attached H. pylori cells. In addition, the H. pylori morphology was changed to a coccoidal shape in the presence of the extracts. In conclusion, the I. britannica extracts were effective against H. pylori strains in vitro, irrespective of genotype status, and could therefore be used as novel functional foods.

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS) were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s)., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

There is considerable interest in alternative approaches to inhibit Helicobacter pylori (H. pylori) and thus treat many stomach diseases. Propolis is a pharmaceutical mixture containing many natural bioactive substances. The aim of this study was to use propolis samples to treat H. pylori. The anti-H. pylori and anti-urease activities of 15 different ethanolic propolis extracts (EPEs) were tested. The total phenolic contents and total flavonoid contents of the EPE were also measured. The agar-well diffusion assay was carried out on H. pylori strain J99 and the inhibition zones were measured and compared with standards. All propolis extracts showed high inhibition of H. pylori J99, with inhibition diameters ranging from 31.0 to 47.0 mm. Helicobacter pylori urease inhibitory activity was measured using the phenol-hypochlorite assay; all EPEs showed significant inhibition against the enzyme, with inhibition concentrations (IC 50 ; mg/mL) ranging from 0.260 to 1.525 mg/mL. The degree of inhibition was related to the phenolic content of the EPE. In conclusion, propolis extract was found to be a good inhibitor that can be used in H. pylori treatment to improve human health.

Biomineralization is a process by which living organisms produce minerals. The extracellular production of these biominerals by microbes has potential for various bioengineering applications. For example, crack remediation and improvement of durability of concrete is an important goal for engineers and biomineral-producing microbes could be a useful tool in achieving this goal. Here we report the isolation, biochemical characterization and molecular identification of Pseudomonas azotoformans, a microbe that produces calcite and which potentially be used to repair cracks in concrete structures. Initially, 38 bacterial isolates were isolated from soil and cements. As a first test, the isolates were screened using a urease assay followed by biochemical tests for the rate of urea hydrolysis, calcite production and the insolubility of calcite. Molecular amplification and sequencing of a 16S rRNA fragment of selected isolates permitted us to identify P. azotoformans as a good candidate for preparation of biotechnological concrete. This species was isolated from soil and the results show that among the tested isolates it had the highest rate of urea hydrolysis, produced the highest amount of calcite, which, furthermore was the most adhesive and insoluble. This species is thus of interest as an agent with the potential ability to repair cracks in concrete.

Infected kidney stones refer to stones that form because of urinary tract infections with urease-producing bacteria, secondarily infected stones of any composition, or stones obstructing the urinary tract leading to pyelonephritis. The mainstay of treatment of infection stones is complete stone removal. Kidney stones that obstruct the urinary tract and cause obstructive pyelonephritis are also frequently referred to as infected stones. Obstructive pyelonephritis is a urologic emergency as it can result in sepsis and even death. Infection stones and obstructive stones causing pyelonephritis are different disease processes, and their workup and management are described separately., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

We present a microfluidic optoelectronic sensor for saliva diagnostics with a potential application for non-invasive early diagnosis of stomach cancer. Stomach cancer is the second most common cause of cancer-related deaths in the world. The primary identified cause is infection by a gram-negative bacterium Helicobacter pylori. These bacteria secrete the enzyme urease that converts urea into carbon dioxide (CO2) and ammonia (NH3), leading to their elevated levels in breath and body fluids. The proposed optoelectronic sensor will detect clinically relevant levels of CO2 and NH3 in saliva that can potentially be used for early diagnosis of stomach cancer. The sensor is composed of the embedded in a microfluidic device array of microwells filled with ion-exchange polymer microbeads doped with various organic dyes. The optical response of this unique highly diverse sensor is monitored over a broad spectrum, which provides a platform for cross-reactive sensitivity and allows detection of CO2 and NH3 in saliva at ppm levels., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen and is the cause of bloody diarrhea and hemolytic-uremic syndrome. The virulence repertoire of EHEC includes the genes within the locus of enterocyte effacement (LEE) that are largely organized in five operons, LEE1 to LEE5, which encode a type III secretion system, several effectors, chaperones, and regulatory proteins. In addition, EHEC also encodes several non-LEE-encoded effectors and fimbrial operons. The virulence genes of this pathogen are under a large amount of posttranscriptional regulation. The small RNAs (sRNAs) GlmY and GlmZ activate the translation of glucosamine synthase (GlmS) in E. coli K-12, and in EHEC they destabilize the 3' fragments of the LEE4 and LEE5 operons and promote translation of the non-LEE-encoded effector EspFu. We investigated the global changes of EHEC gene expression governed by GlmY and GlmZ using RNA sequencing and gene arrays. This study extends the known effects of GlmY and GlmZ regulation to show that they promote expression of the curli adhesin, repress the expression of tryptophan metabolism genes, and promote the expression of acid resistance genes and the non-LEE-encoded effector NleA. In addition, seven novel EHEC-specific sRNAs were identified using RNA sequencing, and three of them--sRNA56, sRNA103, and sRNA350--were shown to regulate urease, fimbria, and the LEE, respectively. These findings expand the knowledge of posttranscriptional regulation in EHEC., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Urease gene expression in Streptococcus salivarius 57.I, a strain of one of the major alkali producers in the mouth, is induced by acidic pH and excess amounts of carbohydrate. Expression is controlled primarily at the transcriptional level from a promoter, pureI. Recent sequencing analysis revealed a CodY box located 2 bases 5' to the -35 element of pureI. Using continuous chemostat culture, transcription from pureI was shown to be repressed by CodY, and at pH 7 the repression was more pronounced than that in cells grown at pH 5.5 under both 20 and 100 mM glucose. The direct binding of CodY to pureI was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP)-quantitative real-time PCR (qPCR). The result of ChIP-qPCR also confirmed that the regulation of CodY is indeed modulated by pH and the binding of CodY at neutral pH is further enhanced by a limited supply of glucose (20 mM). In the absence of CodY, the C-terminal domain of the RNA polymerase (RNAP) α subunit interacted with the AT tracks within the CodY box, indicating that CodY and RNAP compete for the same binding region. Such regulation could ensure optimal urease expression when the enzyme is most required, i.e., at an acidic growth pH with an excess amount of carbon nutrients., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Vibrio parahaemolyticus is a pathogenic marine bacterium that causes food-borne gastroenteritis and, less commonly, wound infections. As is the case for many pathogens, all V. parahaemolyticus strains possess at least one erythrocyte-lysing haemolysin. In addition, many V. parahaemolyticus also possess the enzyme urease. We tested 206 environmental V. parahaemolyticus isolates from Mississippi coastal waters for urease and haemolytic activity using urea agar with added salt and Wagatsuma agar, respectively. The relative abundance of haemolysin-producing V. parahaemolyticus was consistently high throughout the sampling period. In contrast, the number of urease-positive organisms increased from 36% in 2006 to 80% in 2007 and 97% in 2009. We then tested the ability of four strains representing each of the three sample years along with seven other bacterial strains for their ability to grow in seawater urea and raise the pH of this seawater broth. Finally, one of the 4 strains was tested for its ability to form an alkaline microhabitat immediately above its biofilm., Significance and Impact of the Study: The results of this study illustrate that V. parahaemolyticus has the ability to create alkaline microhabitats that could enhance virulence, including virulence from haemolysins. This finding could have both clinical and ecological impact as to how V. parahaemolyticus can modify its habitat., (© 2014 The Society for Applied Microbiology.)

Background: Hyperammonemic encephalopathy may occur when urease-positive bacteria in the urinary tract produce ammonium which directly enters systemic circulation. Predisposing conditions such as a neurogenic bladder can increase both urinary tract infection and urine stagnation., Case Presentation: We describe the case of a 66 years old woman with a neurogenic bladder who twice developed hyperammonemic encephalopathy following urinary tract infection. During the second episode Escherichia coli and Enterococcus faecalis have been isolated in the urine. The neurologic examination showed psychomotor slowing, weak photomotor reflex, nystagmus in the lateral gaze and asterixis. The EEG showed triphasic waves which disappeared along with clinical recovery., Conclusion: Escherichia coli and Enterococcus faecalis are commonly considered urease-negative bacteria. Although frequently involved in urinary tract infections, their role in causing hyperammonemic encephalopathy have not been previously reported. Moreover, despite only one case with a neurogenic bladder have been described so far, our is the first patient with reoccurring hyperammonemic encephalopathy secondary to urinary tract infections.

Objective: To construct the multi epitope prokaryotic expression plasmid and appropriate engineering bacteria expressing the multi-epitope fusion protein of urea membrane channel protein (UreI), urease B subunit (UreB) and adhesin (HpaA) of Helicobacter pylori, then study its microbiological characteristics., Methods: The target sequence contains multi epitope gene sequence of Helicobacter pylori were designed and synthesized, subsequently; it was subcloned into the expression vector pET28a (+), confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein rIBA was expressed in E. coli Rosseta (DE3) and analyzed by Western blot., Results: The plasmid of pET28a(+)/IBA was constructed successfully, confirmed by endonuclease digestion and sequence analyze. The expressed rIBA protein with relative molecular mass about 40 x 10(3) and can be detected by Western blot., Conclusion: The prokaryotic engineering bacteria expression multi-epitope of the Helicobacter pylori was constructed successfully. The recombinant protein rIBA expressed by the engineering bacteria can be identified by Sydney strain 1 of Helicobacter pylori (H. pylori SS1) specific antibody IgY, which demonstrated that the rIBA has high correlation with H. pylori SS1.

This paper describes the isolation of a potent extracellular urease producing microorganism, identified by 16S rRNA as Arthrobacter creatinolyticus MTCC 5604 and its medium optimization by classical one-factor-at-a-time method and central composite rotatable design (CCRD), a tool of response surface methodology (RSM). An optimal activity of 9.0 U ml(-1) was obtained by classical method and statistical optimization of the medium resulted in an activity of 17.35 U ml(-1) at 48 h and 30 °C. This activity was 4.91 times greater than the initial activity (3.53 U ml(-1) ) from the basal medium and the enzyme showed maximum activity at pH 8.0 and 60 °C and was stable at pH 7.0-9.0 and temperatures up to 50 °C. Furthermore, the enzyme was assessed for its activity reduction by determining the inhibitory concentration (IC50 ) of heavy metal ions and the inhibition of urease was in the order of Cu(II) > Cd(II) > Zn(II) > Ni(II). Urease was highly sensitive to Cu(II) and its inhibition was 94% and 100% in model solutions containing a mixture of Cu(II) with heavy metal ions Cd(II) and Zn(II), respectively. The results of these studies suggested that the enzyme could be utilized as sensors to determine the levels of Cu(II) ions in industrial effluents, contaminated soil and ground water., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin.

Background: Intestinal microbiome constitutes a symbiotic ecosystem that is essential for health, and changes in its composition/function cause various illnesses. Biochemical milieu shapes the structure and function of the microbiome. Recently, we found marked differences in the abundance of numerous bacterial taxa between ESRD and healthy individuals. Influx of urea and uric acid and dietary restriction of fruits and vegetables to prevent hyperkalemia alter ESRD patients' intestinal milieu. We hypothesized that relative abundances of bacteria possessing urease, uricase, and p-cresol- and indole-producing enzymes is increased, while abundance of bacteria containing enzymes converting dietary fiber to short-chain fatty acids (SCFA) is reduced in ESRD., Methods: Reference sets of bacteria containing genes of interest were compiled to family, and sets of intestinal bacterial families showing differential abundances between 12 healthy and 24 ESRD individuals enrolled in our original study were compiled. Overlap between sets was assessed using hypergeometric distribution tests., Results: Among 19 microbial families that were dominant in ESRD patients, 12 possessed urease, 5 possessed uricase, and 4 possessed indole and p-cresol-forming enzymes. Among 4 microbial families that were diminished in ESRD patients, 2 possessed butyrate-forming enzymes. Probabilities of these overlapping distributions were <0.05., Conclusions: ESRD patients exhibited significant expansion of bacterial families possessing urease, uricase, and indole and p-cresol forming enzymes, and contraction of families possessing butyrate-forming enzymes. Given the deleterious effects of indoxyl sulfate, p-cresol sulfate, and urea-derived ammonia, and beneficial actions of SCFA, these changes in intestinal microbial metabolism contribute to uremic toxicity and inflammation.

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 micromol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 micromol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease alpha (A) and beta (B), raised against Helicobacter pylori.

Corynebacterium propinquum is a Gram-positive rod occasionally recovered from clinical infections which, according to 16S rRNA gene sequencing, is most closely related (>99% sequence similarity) to Corynebacterium pseudodiphtheriticum. The two species are very similar biochemically, commonly differentiated by a single test, the detection of urease, where strains of C. propinquum are described as being urease-non-producing and strains of C. pseudodiphtheriticum are described as urease-producing. In this study, historical and contemporary strains of C. propinquum and C. pseudodiphtheriticum from this laboratory were definitively characterized, which included use of rpoB sequencing. Urease-producing strains of C. propinquum as well as typical urease-non-producing isolates were identified after rpoB sequencing, with six of these being originally identified as C. pseudodiphtheriticum. Based on these observations, we propose emendation of the description of C. propinquum to include strains which produce urease. MALDI-TOF analysis may be a useful tool to differentiate these taxa. Existing commercial databases should be updated to include urease-positive strains of C. propinquum.

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview.

A set of 25 urease-producing, yellow-pigmented enterococci was isolated from environmental sources. Phenotypic classification divided the isolates into two phena. Both phena were characterized using 16S rRNA gene sequence analysis, DNA base composition, rep-PCR fingerprinting and automated ribotyping. The obtained data distinguished the isolates from all members of the genus Enterococcus with validly published names and placed them in the Enterococcus faecalis species group. DNA-DNA hybridization experiments, pheS and rpoA sequencing and whole-cell protein electrophoresis provided conclusive evidence for the classification of each phenon as a novel species of the genus Enterococcus, for which the names Enterococcus ureilyticus sp. nov. (type strain CCM 4629(T)  = LMG 26676(T)  = CCUG 48799(T)), inhabiting water and plants, and Enterococcus rotai sp. nov. (type strain CCM 4630(T)  = LMG 26678(T)  = CCUG 61593(T)), inhabiting water, insects (mosquitoes) and plants, are proposed.

Trichophyton rubrum var. raubitschekii is a rare anthropophilic dermatophyte isolated around the world from tinea corporis, tinea cruris, tinea pedis and tinea unguium. In this study, the isolation rate of T. rubrum var. raubitschekii was studied in 200 cases of tinea pedis and tinea unguium in Japan. The 200 clinical isolates were shown to be of downy type as their colonies on Sabouraud's dextrose agar were white to cream, suede-like to downy, with a yellow-brown to wine-red reverse, and they produced few macroconidia. The type strain of T. rubrum var. raubitschekii (CBS 100084) and one clinical isolate (KMU 8337; isolated at Kanazawa) of downy type tested positive for urease, but the reference strain of T. rubrum (CBS 392.58) and the remaining 199 clinical isolates tested negative. Further epidemiological investigations are required to study human cases of infection with the granular type of T. rubrum and T. rubrum var. raubitschekii in Japan., (© 2012 Japanese Dermatological Association.)

Objectives: To evaluate the effect of plum juice on struvite calculus formation in vitro and to explore the effect of plum juice on urease-producing bacteria and urease activity. The compliance of available drugs is low for struvite calculus after surgical treatment and functional food may represent a good choice as an alternative therapy., Materials and Methods: Antibacterial activity was assessed using a microdilution antimicrobial susceptibility test. Urease activity was determined by measuring ammonia production. Struvite crystals were induced by Proteus mirabilis in artificial urine with natural and pH-adjusted plum juice. The optical density (OD)(600) and pH of artificial urine were examined, as well the shape and weights of crystals., Results: Natural plum juice showed an antibacterial effect on urease-producing bacteria, whereas the pH-adjusted juice did not. A concentration-dependent inhibition on urease activity was found for both natural and pH-adjusted juice. Natural plum juice at a high concentration of 0.5% showed an obvious inhibition on the increase of OD(600) and pH of the artificial urine, and crystal formation was prevented by up to or more than 8 h, depending on the concentration of juice. Crystal weight in the natural plum juice groups was decreased in a concentration-dependent manner. The pH-adjusted plum juice did not show any effect on OD(600) and pH, although the presence of juice changed the crystal habit, indicating that the juice slowed the growth rate of crystals., Conclusions: Natural plum juice at high and moderate concentrations prevented the formation of P. mirabilis-induced crystals for up to 8 h in artificial urine. Although pH-adjusted and low-concentration natural juice did not prevent the occurrence of crystals, both types of juice slowed their growth rate.

Genes encoding the enzyme urease were integrated in a Thermoanaerobacterium saccharolyticum ethanologen. The engineered strain hydrolyzed urea, as evidenced by increased cellular growth and elevated final pH in urea minimal medium and urease activity in cell free extracts. Interestingly, replacement of ammonium salts with urea resulted in production of 54 g/L ethanol, one of the highest titers reported for Thermoanaerobacterium. The observed increase in ethanol titer may result from reduced pH, salt, and osmolality stresses during fermentation. Urea utilization is attractive for industrial scale fermentation, where pH control is technically challenging and increased ethanol titer is desirable., (Copyright © 2012. Published by Elsevier Inc.)

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

Background: Nontypeable Haemophilus influenzae is a common cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Prior studies have shown that H. influenzae expresses abundant urease during growth in the middle ear of the chinchilla and in pooled human sputum, suggesting that expression of urease is important for colonization and infection in the hostile environments of the middle ear and in the airways in adults. Virtually nothing else is known about the urease of H. influenzae, which was characterized in the present study., Results: Analysis by reverse transcriptase PCR revealed that the ure gene cluster is expressed as a single transcript. Knockout mutants of a urease structural gene (ureC) and of the entire ure operon demonstrated no detectable urease activity indicating that this operon is the only one encoding an active urease. The ure operon is present in all strains tested, including clinical isolates from otitis media and COPD. Urease activity decreased as nitrogen availability increased. To test the hypothesis that urease is expressed during human infection, purified recombinant urease C was used in ELISA with pre acquisition and post infection serum from adults with COPD who experienced infections caused by H. influenzae. A total of 28% of patients developed new antibodies following infection indicating that H. influenzae expresses urease during airway infection. Bacterial viability assays performed at varying pH indicate that urease mediates survival of H. influenzae in an acid environment., Conclusions: The H. influenzae genome contains a single urease operon that mediates urease expression and that is present in all clinical isolates tested. Nitrogen availability is a determinant of urease expression. H. influenzae expresses urease during human respiratory tract infection and urease is a target of the human antibody response. Expression of urease enhances viability in an acid environment. Taken together, these observations suggest that urease is important for survival and replication of H. influenzae in the human respiratory tract.

Background: We hypothesize that pH difference between acid-secreting corpus and non-secreting antrum might influence the activity of H. pylori's urease and/or related genes. We therefore measured urease activity and the expression of amiE whose encoded protein that hydrolyzes short-chain amides to produce ammonia., Materials and Methods: Fifty-four patients were recruited into this study. Each gastroscopic biopsy specimen collected from the antrum and body of each patient was immediately used to measure urease activity using serial changes of urease activity (ammonia levels) during 60 minutes. Probe specific for amiE was labeled with a biotin nick-translation kit and was used to detect expression of these genes (mRNA) in fresh-frozen gastroscopic biopsy specimens using fluorescent in situ hybridization (FISH)., Results: Urease activity at 60 minutes from the gastric antrum and body of all patients infected with H. pylori was 399.5 ± 490.5 and 837.9 ± 1038.9 μg/dL, respectively (p = .004). Urease activity in the antrum was correlated with H. pylori density. Urease activity or H. pylori density in the antrum was significantly correlated with chronic active inflammation; in contrast, this correlation was not found in the gastric body. The expression level of amiE was 1.5 times higher (p < .05) in the gastric body compared with the antrum., Conclusion: Topographically, the urease activity in body was much higher than in antrum. The expression level of amiE was higher in the gastric body compared with the antrum., (© 2011 Blackwell Publishing Ltd.)

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, beta-galactosidase activity was 4.4-fold lower after 30 degrees C cultivation than after 37 degrees in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either 30 degrees or 37 degrees by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

Pathogenicity islands (PAIs) are a specific group of genomic islands that contribute to genomic variability and virulence of bacterial pathogens. Using a strain-specific comparative genomic hybridization array, we report the identification of a 94-kb PAI, designated ICEPm1, that is common to Proteus mirabilis, Providencia stuartii, and Morganella morganii. These organisms are highly prevalent etiologic agents of catheter-associated urinary tract infections (caUTI), the most common hospital acquired infection. ICEPm1 carries virulence factors that are important for colonization of the urinary tract, including a known toxin (Proteus toxic agglutinin) and the high pathogenicity island of Yersinia spp. In addition, this PAI shares homology and gene organization similar to the PAIs of other bacterial pathogens, several of which have been classified as mobile integrative and conjugative elements (ICEs). Isolates from this study were cultured from patients with caUTI and show identical sequence similarity at three loci within ICEPm1, suggesting its transfer between bacterial genera. Screening for the presence of ICEPm1 among P. mirabilis colonizing isolates showed that ICEPm1 is more prevalent in urine isolates compared to P. mirabilis strains isolated from other body sites (P<0.0001), further suggesting that it contributes to niche specificity and is positively selected for in the urinary tract.

The problem of catheter encrustation stems from infection by urease-producing bacteria. These organisms generate ammonia from urea, elevate the pH of urine and cause crystals of calcium and magnesium phosphates to form in the urine and the biofilm that develops on the catheter. In this study, a laboratory model was used to compare the ability of 12 urease-positive species of urinary tract pathogens to encrust and block catheters. Proteus mirabilis, Proteus vulgaris and Providencia rettgeri were able to raise the urinary pH above 8.3 and produce catheter-blocking crystalline biofilms within 40 h. Morganella morganii and Staphylococcus aureus elevated the pH of urine to 7.4 and 6.9, respectively, and caused some crystal deposition in the biofilms but did not block catheters in the 96 h experimental period. Isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Serratia marcescens, Pseudomonas aeruginosa and Providencia stuartii were only capable of raising the pH of urine to a maximum of 6.4 and failed to cause crystal deposition in the biofilm. The most effective way to prevent catheter encrustation was shown to be diluting urine and increasing its citrate concentration. This strategy raises the nucleation pH (pH(n)) at which calcium and magnesium phosphates crystallize from urine. Increasing the fluid intake of a healthy volunteer with citrated drinks resulted in urine with a pH(n) of >8.0 in which catheter encrustation was inhibited. It is suggested that this dietary strategy will be an effective means of controlling catheter encrustation, whichever bacterial species is causing the problem.

Yersinia pseudotuberculosis is an enteric bacterium which must overcome the acidic stress in host organs for successful colonization, but how this bacterium survives in acidic conditions remains largely unknown. In the present study, the importance of OmpR in acid survival of Y. pseudotuberculosis YpIII was confirmed by the fact that mutation of ompR (strain DeltaompR) greatly reduced cell survival at pH 4.5 or lower. To characterize the regulatory role of OmpR in this acid survival process, proteomic analysis was carried out to compare YpIII at pH 7.0 and pH 4.5 with DeltaompR at pH 7.0, and urease components were revealed to be the main targets for OmpR regulation. Addition of urea to the culture medium also enhanced acid survival of YpIII but not DeltaompR and urease activity was significantly induced by acid in YpIII but not in DeltaompR. Each of the seven components of the YpIII urease gene cluster was fused to a lacZ reporter and their expression was dramatically decreased in a DeltaompR background; this supports the notion that OmpR positively regulates urease expression. Furthermore, gel shift analysis revealed that OmpR binds to the deduced promoter regions of three polycistronic transcriptional units (ureABC, ureEF and ureGD) in the urease cluster, suggesting that the regulation of OmpR to urease components is direct. Taken together, these data strongly suggest that OmpR activates urease expression to enhance acid survival in Y. pseudotuberculosis.

Phenotypic mutants of Sporosarcina pasteurii (previously known as Bacillus pasteurii) (MTCC 1761) were developed by UV irradiation to test their ability to enhance urease activity and calcite production. Among the mutants, Bp M-3 was found to be more efficient compared to other mutants and wild-type strain. It produced the highest urease activity and calcite production compared to other isolates. The production of extracellular polymeric substances and biofilm was also higher in this mutant than other isolates. Microbial sand plugging results showed the highest calcite precipitation by Bp M-3 mutant. Scanning electron micrography, energy-dispersive X-ray and X-ray diffraction analyses evidenced the direct involvement of bacteria in CaCO3 precipitation. This study suggests that calcite production by the mutant through biomineralization processes is highly effective and may provide a useful strategy as a sealing agent for filling the gaps or cracks and fissures in any construction structures.

In the development of an oral vaccine against Helicobacter pylori, H. pylori urease subunit B (UreB) was expressed in a food-grade delivery vehicle, Lactococcus lactis NZ3900. The ureB gene (Genbank accession no. FJ436980) was amplified by polymerase chain reaction (PCR) from MEL-Hp27. The PCR-amplified ureB gene was cloned in the E. coli-L. lactis shuttle vector pNZ8110 and transformed into E. coli MC1061. After the transformant had been identified, the recombinant plasmid was purified and electrotransformed into L. lactis NZ3900. The conditions of UreB expression in the L. lactis transformant were optimized by orthogonal experiment. The maltose binding protein (MBP)-UreB fusion protein expressed by TB(1)/pMAL-c2X-ureB was used to cultivate mice polyclonal anti-UreB serum after purification by the amylose prepacked column. The Western blot method was adopted to confirm whether the UreB expressed by L. lactis transformant had immunoreactivity. The optimized conditions for UreB expression were as follows. Nisin 40 ng/ml was added to the medium when the recombinant grew to OD(600) approximately 0.30-0.40 and the induction time lasted 5 h. As a result, the maximum yield of UreB was 27.26 microg/mL of medium, and the maximum percentage of UreB in cell extracts of the L. lactis transformant reached its peak at 20.19%. Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity. All these results make an appealing case for construction of the food-grade vaccine for H. pylori.

Purpose: Helicobacter pylori is the etiological agent in duodenal and peptic ulcers. The growing problem of antibiotic resistance by the organism demands the search for novel compounds, especially from natural sources. This study was conducted to evaluate the effect of Camellia sinensis extracts on the urease enzyme that is a major colonization factor for H. pylori., Methods: Minimum inhibitory concentrations of nonfermented and semifermented C. sinensis methanol: water extracts were assessed by broth dilution method. Examination of the urease function was performed by Mc Laren method, and urease production was detected on 12% SDS polyacrylamide gel electrophoresis from whole cell and membrane bound proteins., Results: Both extracts had inhibitory effects against H. pylori and urease production. At a concentration of 2.5 mg/ml of nonfermented extract and 3.5 mg/ml of semifermented extract the production of Ure A and Ure B subunits of the urease enzyme were inhibited completely. A concentration of 4 mg/ml of nonfermented and 5.5 mg/ml of semifermented extract were bactericidal for H. pylori., Conclusions: C. sinensis extracts, especially the nonfermented, could reduce H. pylori population and inhibit urease production at lower concentrations. The superior effect of nonfermented extract is due to its rich polyphenolic compounds and catechin contents.