Absence Of Urease Research Articles

Enzyme cum pH dual-responsive controlled release of avermectin from functional polydopamine microcapsules

Novel enzyme cum pH dual-responsive microcapsule were prepared with an aim to enhance the utilization of pesticides and reduce environmental pollution. The synthetic procedure involved the introduction of urea bonds by forming a covalent bond between isocyanate–functionalized polydopamine (PDA) microcapsules and polyethyleneimine (PEI). The prepared AVM@PDA-IPTS-PEI microcapsules could effectively protect avermectin (AVM) from ultraviolet radiation, and thus decelerate the decomposition rate; the cumulative release showed positive correlations with pH and the urease activity. The AVM release rate was highest at pH 10. At pH 7, the release rate was enhanced after addition of urease, showing improved urease-responsive property under neutral or weak base condition. Especially at pH 7 in presence of urease, the release behavior was in agreement with the Logistic model and able to describe the S-shaped/sigmoidal release profiles, whereas Korsmeyer–Peppas model was better suited in the absence of urease, as the value of K1 was smaller than 0.45, suggesting that the behavior was controlled by Fick’s diffusion. AVM@PDA-IPTS-PEI displayed better adhesion property than AVM, and the microcapsule ameliorated foliage wettability. AVM@PDA-IPTS-PEI exhibited the insecticidal property similarly with AVM, which can be used for reducing the applied quantity of pesticides and improving their efficacy.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

Ubiquitous urease affects soybean susceptibility to fungi

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9894-1) contains supplementary material, which is available to authorized users.

Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine

A novel Gram-positive, motile, endospore-forming, aerobic bacterium was isolated from the NASA Phoenix Lander assembly clean room that exhibits 100 % 16S rRNA gene sequence similarity to two strains isolated from a deep subsurface environment. All strains are rod-shaped, endospore-forming bacteria, whose endospores are resistant to UV radiation up to 500 J m(-2). A polyphasic taxonomic study including traditional phenotypic tests, fatty acid analysis, 16S rRNA gene sequencing and DNA-DNA hybridization analysis was performed to characterize these novel strains. The 16S rRNA gene sequencing convincingly grouped these novel strains within the genus Paenibacillus as a separate cluster from previously described species. The similarity of 16S rRNA gene sequences among the novel strains was identical but only 98.1 to 98.5 % with their nearest neighbours Paenibacillus barengoltzii ATCC BAA-1209(T) and Paenibacillus timonensis CIP 108005(T). The menaquinone MK-7 was dominant in these novel strains as shown in other species of the genus Paenibacillus. The DNA-DNA hybridization dissociation value was <45 % with the closest related species. The novel strains had DNA G+C contents of 51.9 to 52.8 mol%. Phenotypically, the novel strains can be readily differentiated from closely related species by the absence of urease and gelatinase and the production of acids from a variety of sugars including l-arabinose. The major fatty acid was anteiso-C(15 : 0) as seen in P. barengoltzii and P. timonensis whereas the proportion of C(16 : 0) was significantly different from the closely related species. Based on phylogenetic and phenotypic results, it was concluded that these strains represent a novel species of the genus Paenibacillus, for which the name Paenibacillus phoenicis sp. nov. is proposed. The type strain is 3PO2SA(T) ( = NRRL B-59348(T) = NBRC 106274(T)).

The role of Klebsiella pneumoniae urease in intestinal colonization and resistance to gastrointestinal stress

The first step in nosocomial infections due to Klebsiella pneumoniae is colonization of the patient's gastrointestinal (GI) tract. In a previous work, signature-tagged mutagenesis was used in a murine model to identify 13 genes required for efficient colonization, two of which were involved in urea metabolism. The role of urease was further investigated by the construction and analysis of an isogenic urease-deficient mutant. The behavior of both the wild-type strain and the urease-deficient mutant was tested under hostile conditions, reproducing stresses encountered in the GI environment. The wild-type strain had an acid tolerance response (ATR) to inorganic acid, was resistant to organic acids (38.5% survival) and was able to survive concentrations of bile encountered in vivo. The absence of urease did not affect the resistance of K. pneumoniae to acid and bile stresses, but the enhanced adhesion response to Int-407 cells after exposure to bile observed with the wild-type strain was no longer detected with the urease mutant. When tested in the murine intestinal colonization model, both strains were mainly recovered in the large intestine parts, and the mutant was impaired in its colonization capacities, but only when tested in competition with the wild-type strain. These findings emphasize the prominent role played by metabolic function in the colonization process of such a complex ecosystem as the host GI tract.

How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in-vitro studies.

Survival of Helicobacter pylori is dependent upon urease in the cytoplasm and at the bacterial surface. We have sought to clarify how alkaline ammonium salts, released from urea by this enzyme, might alter mucus pH and so affect growth and motility of the bacterium in the gastric mucus environment. Experiments were conducted in vitro to determine how the growth and motility of H. pylori are affected by changes in external pH, and how the bacterium, by hydrolysing urea, alters the pH of the bicarbonate buffer that occurs at the gastric mucosal surface. These data were fitted into experimental models that describe how pH varies within the mucus layer in the acid-secreting stomach. H. pylori was motile between pH 5 and 8, with optimal motility at pH 5. It grew between pH 6 and 8, with optimal growth at pH 6. The bacterium had urease activity between pH 2.7 and 7.4, as evidenced by pH rises in bicarbonate-buffered solutions of urea. Changes in buffer pH were dependent upon initial pH and urea concentration, with the greatest rate of pH change occurring at pH 3. Modelling experiments utilizing these data indicated that (1) in the absence of urease, H. pylori growth and motility in the mucus layer would be restricted severely by low mucus pH in the acid-secreting stomach, and (2) urease will sometimes inhibit H. pylori growth and motility in the mucus layer by elevating the pH of the mucus environment above pH 8. Urease is essential to the growth and motility of H. pylori in the mucus layer in the acid-secreting stomach, but, paradoxically, sometimes it might suppress colonization by raising the mucus pH above 8. This latter effect may protect the bacteria from the adverse consequences of overpopulation.

In vitro short-term study of ammonium–nitrogen production from cattle urine: influence of ampicillin, hydroquinone and animal litter materials

The production of NH+4-N following in vitro incubation of cattle urine was monitored for 24 h in the presence of ampicillin sodium salt (0, 32, 64, 128 mg/l), hydroquinone (0, 16·7, 33·4, 66·8 mg/l), wheat straw (0, 3·3, 6·6, 13·2 g/l) or spruce sawdust (0, 3·3, 6·6, 13·2 g/l) with (20 IU) or without urease. Each concentration of ampicillin, hydroquinone, wheat straw or spruce sawdust was tested in triplicate.The equipment consisted of Woulff flasks containing 300 ml of a buffered solution (0·02 M; pH 7·50) with 1 ml of cattle urine. The cattle urine was characterized by measuring the main nitrogen contents, which were 6·52 mg total-N/ml, 5·96 mg urea-N/ml and 0·026 mg ammonium-N/ml. The initial pH of urine was 7·84. Ammonium and nitrate concentrations, and pH were monitored at zero- time and after 3, 6 and 24 h of incubation with the cattle urine.The addition of urease to the flasks containing urine induced a significant increase in the production of ammonium-N, from 1·83 to 6·32 mg NH+4-N/flask after 24 h of incubation.In the presence of urease, an inhibitory effect was recorded in NH+4-N production with ampicillin and spruce sawdust. In contrast, hydroquinone inhibited urease activity and wheat straw adsorbed the NH+4-N produced, both causing a dose-dependent relationship.In the absence of urease, ampicillin, hydroquinone, wheat straw or spruce sawdust caused a dose- related decrease in NH+4-N production. However, the highest amounts of wheat straw (6·6 and 13·2 g/flask) exhibited a temporary increase in NH+4-N production during the first 6 h. This is probably due to a generation of extra NH+4-N as compared to the control flasks without straw. However, at 24 h, the situation paralleled the other materials.Although pH increased approximately from 7·50 to 7·65 during the hydrolysis of urea in the presence of urease, no gaseous ammonia was volatilized into a 0·1 M HCl flask. Moreover, no increase in nitrate concentration was found during the incubation.The present results suggest that ampicillin, hydroquinone and spruce sawdust could be used in order to reduce NH+4-N production from cattle urine. The NH+4-N already produced could probably be absorbed on wheat straw.

Characterizing acidification kinetics by measuring pH and electrical conductivity in batch thermophilic lactic fermentations

On-line measurements of pH and electrical conductivity data have made it possible to access time and rate feature points of thermophilic lactic acid fermentations. Ten feature points characterize curves of acidification and conductivity changes using the main points of inflection observed. The presence or absence of urease also changes the observed kinetics and corresponding feature points. These phenomena and the time patterns of the biomass and products permit an understanding of the meanings of the feature points. These points showed the excellent reproducibility of fermentations conducted in standard conditions, with coefficients of variation lower than 5.1% for nine of them. They were also used to compare the effects of the type of starter, temperature and culture medium. The temperature affects the urease activity and acidification optima. The presence of fat (32 g/l) in the medium does not change any feature point, while the presence of sucrose (90 g/l) results in a decrease in the acidification rate and a longer fermentation time.

The inheritance of a urease-null trait in soybeans

Four soybean seed urease nulls (lacking both the activity and antigen of the embryo-specific urease) were intermated and the F1 and F2 seed examined for urease activity. Both generations were without urease activity, and the nulls were therefore considered noncomplementing. In crosses of each null line to cultivars homozygous for the allelic, codominantly inherited urease slow or fast isozyme, the F1 seed expressed the embryo-specific urease isozyme of the urease-expressing parent. A 3 ∶ 1 segregation for presence and absence of urease was observed in progeny from F1 and heterozygous F2 plants. The F2 and F3 from fastXnull combinations revealed that urease-positive seed were all phenotypically urease fast, while the same seed from slowXnull combinations showed a segregation of one seed containing a fast urease, either exclusively or in a heterozygous state with the slow isozyme, for every 69 phenotypic slows. Data pooled from F2 plants which segregate for both the presence (Sun) and absence (Sun) of urease and for the fast (Eu1-b) or slow (Eu1-a) urease allele indicate that the null lesion (Sun) is linked to Eu1 by approximately one map unit. The evidence is consistent with two models: (1) sun is an allele at the embryo-specific urease isozyme locus (Eu1) and that a high degree of exchange (and/or conversion) within the locus results in a 1% recombination frequency between the null trait and urease allozyme; (2) sun is at a distinct locus which is separated by one map unit from the embryo-specific urease isozyme locus (Eu1) upon which it acts in the cis position. Polyadenylated embryo RNA from one of the null lines, PI 229324, exhibited no urease template activity in vitro. Thus, the lack of urease antigen is due to lack of accumulation of translatable urease mRNA. The availability of soybeans lacking seed urease should be extremely useful to breeders as a trait for linkage studies and to geneticists as a transformation marker.