Factors affecting growth and urease production by Trichophyton spp.

Among Trichophyton spp. examined for urease production, T. rubrum was negative, whereas T. mentagrophytes appeared to be the most active species. Urease was not detected in cell-free culture fluids of the tested fungi. The endocellular urease of the test fungi was essentially constitutive. Moreover, addition of urea to the growth medium of these organisms markedly inhibited their mycelial biomass and ureolytic yield. Environmental factors showed variable effects on the test fungi and there was no correlation between mycelial growth and urease activity of these fungi.     

Urea as sole source of nitrogen for plant growth

Summary Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 grams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.     

Studies on the phospholipase A of dermatophytes

The presence of phospholipase A activity was detected in three dermatophytes:Microsporum cookei, Trichophyton mentagrophytes, andEpidermophyton floccosum. The activity was always higher inT. mentagrophytes than inM. cookei andE. floccosum. All exhibited phospholipase A₁ and A₂ activities, but the activity was largely A₂ inM. cookei and A₁ inE. floccosum. T. mentagrophytes possessed almost equal activities of phospholipase A₁ and A₂.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Factors affecting ureolytic activities ofPenicillium waksmanii isolated from sewage sludge

Twenty one fungal isolates belonging to 7 genera were screened for ureolytic activity. APenicillium waksmanii isolate was found to be the most potent and was selected for further study. No ammonia-nitrogen was detected inP. waksmanii cultures either urea-free or containing up to 1 g urea per L. The maximum extracellular urease production was recorded at a urea concentration of 15 g/L. It peaked after 6 d of incubation at 25°C when the initial pH of the glucose—peptone broth was adjusted to 6. On the other hand, the highest fungus biomass was detected at a concentration of 2 g urea per L after 4 d of incubation at 35°C when the pH of the medium was 8. The intracellular urease activity (measured in cell-free extract) was the highest at 12 mg urea per mL after 75-min incubation at 25°C at pH 8. Incubation temperature of 25°C favored both urease production and activity.     

Pleiotropic soybean mutants defective in both urease isozymes

Summary Two new soybean [Glycine max (L.) Merr. cv. Williams] loci, designated Eu2 and Eu3, were identified in which ethyl methanesulfonate (EMS)-induced mutation eliminated urease activity. These loci showed no linkage to each other or to the Sun-Eul locus described in the accompanying paper (Meyer-Bothling and Polacco 1987). Unlike sun (seed urease-null) mutations those at Eu2 and Eu3 affected both urease isozymes: the embryo-specific (seed) and the ubiquitous (leaf) urease. The eu2/eu2 mutant had no leaf activity and 0.6% normal seed activity. Two mutant Eu3 alleles were recovered, eu3-e1 and Eu3-e3. The eu3-e1/eu3-e1 genotype lacked both activities while Eu3-e3/Eu3-e3 had coordinately reduced leaf (0.1%) and seed (0.1%) activities. Only the Eu3-e3 mutation showed partial dominance, yielding about 5%–10% normal activity for each urease in the heterozygous state. Each homozygous mutant contained normal levels of embryo-specific urease mRNA and protein subunit, both of normal size. However, urease polymerization was aberrant in all three mutants. In all cases where urease could be measured, it was found to be temperature sensitive and, in addition, the embryospecific urease of Eu3-e3/Eu3-e3 had an altered pH dependence. These mutants may be defective in a urease maturation function common to both isozymes as suggested by the normal levels of urease gene product, coordinately (or nearly so) reduced urease isozyme activities, temperature sensitivity in both ureases (Eu3-e3) and the non-linkage of Eu2 and Eu3 to the locus encoding embryo-specific urease (Sun-Eul). Ubiquitous urease activity is reduced in mutant seed coat and callus culture as well as in leaf and cotyledon tissue. No mutant callus utilized urea (5 to 10 nM) as sole nitrogen source. However, all mutant cell lines tolerated normally toxic levels of urea (25 to 250 mM) added to medium containing KNO₃/NH₄NO₃ as nitrogen source. Urea thus may be used in cell culture as a selection agent for phenotypes either lacking or regaining an active ubiquitous urease.     

Biolarvicidal activity of extracellular metabolites of the keratinophilic fungus <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis> against arvae of <Emphasis Type="Italic">Aedes aegypti</Emphasis> — a major vector for Chikungunya and dengue

The larvicidal activity of extracellular metabolites of keratinophilic fungus Trichophyton mentagrophytes against Aedes aegypti larvae was determined. T. mentagrophytes was isolated from soil by the feather baiting technique. Culture filtrates (10–100 μL/mL) were found to be entomotoxic to 3rd instars larvae of A. aegypti (L3), LC₅₀ and LC₉₀ being 110 ± 11.5 and 200 ± 20.7 μL/mL, respectively, after 2 d. Extracellular metabolites are proteinaceous in nature and more specific to chitin of mosquito larvae. They degraded cock feather causing an average of 20.0 ± 2.6 % loss in feather mass. Culture filtrate at 100 μL/mL produced 90 % mortality against L3 after 3 d; mortality was increased in dose- and time-dependent manner. These extracellular metabolites of T. mentagrophytes could be regarded as alternatives to synthetic insecticides.     

Urease from Arthrobacter oxydans,a nickel-containing enzyme

In Arthrobacter oxydans, Klebsiella aerogenes and Sporosarcina ureae, growth with urea as a nitrogen source turned out to be more sensitive to inhibition by EDTA than that with ammonia. The inhibition was overcome by added nickel chloride, but not by other divalent metal ions tested. In A. oxydans the uptake of ⁶³Ni was paralleled by an increase in urease (urea amidohydrolase, EC 3.5.1.5) activity under certain conditions. Following growth with radioactive nickel, urease from this strain was enriched by heat treatment and acetone fractionation. Copurification of ⁶³Ni and urease was observed during subsequent Sephadex gel chromatography. Almost the entire labelling was detected together with the purified enzyme after focusing on polyacrylamide gel. The relative molecular mass of the purified urease was estimated to be 242,000. The pH optimum was 7.6, the K _m-value 12.5 mmol/l and the temperature optimum 40°C; heat stability was observed up to 65°C. In presence of 10 mmol/l EDTA the protein-nickel binding remained intact at pH 7; at pH 5 and below, nickel was irreversibly removed with concommitant loss of enzyme activity. The results demonstrated that nickel ions are required for active urease formation in the bacterial strains studied, and that urease from A. oxydans is a nickel-containing enzyme.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Immobilization of watermelon (<Emphasis Type="Italic">Citrullus vulgaris</Emphasis>) urease in agarose gel for urea estimation

Urease from dehusked seeds of watermelon was immobilized in 1.5% agarose gel with 53.9% entrapment. There was negligible leaching (<10% at 4°C) and the same gel membrane could repeatedly be used for seven days. The immobilization exhibited no apparent change in the optimum pH but there was a significant decrease in the optimum temperature (50°C as compared to 65°C for soluble urease). The immobilized urease revealed an apparentK _m of 9.3±0.3 mM; 1.2 times lower than the soluble enzyme (11.4±0.2 mM). Unlike soluble enzyme which was inhibited at 200 mM urea, the immobilized urease was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2 M urea. The time-dependent thermal inactivation kinetics at 48 and 52°C was found to be biphasic, in which half of the initial activity was destroyed more rapidly than the remaining half. These gel membranes were also used for estimating the urea content of the blood samples from the University hospital. The results obtained matched well with those obtained by the usual method employed in the clinical pathology laboratory. The significance of these observations is discussed.     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Characterization of urease from Sporosarcina ureae

Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 μmol urea degraded min^-1 mg protein^-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M _r=65000], the S. ureae enzyme is comprised of three subunits [apparent M _r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.     

Field detection of urease and carbonic anhydrase activity using rapid and economical tests to assess microbially induced carbonate precipitation

Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease-mediated ureolysis and CO₂ (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH₃) and CA (CO₂). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO₃ precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on-site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.     

Exocellular enzyme activity of dermatophytes and other fungi isolated from ruminants in Southern Iraq

Sixteen fungal species were isolated from 182 specimens collected from four ruminants (buffalo, camel, cattle and sheep) in Southern Iraq. Fungi represented by five species of dermatophytes and eleven species of other fungi were screened for the activity of four enzymes; keratinase, proteinase, lipase and amylase. Keratinase was found to be produced by all of the dermatophytes and non-dermatophytes, except for Paecillomyces variottii and Scytalidium lignicola. However, high keratinase activity was expressed by the dermatophytic species particularly by Trichophyton mentagrophytes var. erinacei and Microsporum gypseum. Three dermatophytes viz. M. gypseum, T. verrucosum and T. mentagrophytes var. nodulare were capable of producing protease, lipase and amylase. Although, T. mentagrophytes var. erinacei showed high protease activity, it did not produce lipase and amylase. On the contrary most of the non-dermatophytic species revealed protease and lipase activities higher than the dermatophytes. The Curvularia spp. isolates showed the highest protease and amylase activity, while Aspergillus parasiticus revealed the highest activity of lipase and amylase. No correlation was observed between enzyme activity and the growth rate of the examined fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for carrier-mediated,energy-dependent uptake of urea in some bacteria

Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of ¹⁴C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The K _m for urea uptake was 15–20 times lower (38 M and 13 M urea for A. eutrophus and K. pneumoniae, respectively) than the K _m of urease for urea (650 M and 280 M urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenole     

Amidase and urease activities in plants

Summary Foliar fertilization has received considerable attention in recent years. Because of the importance of amides and urea as N sources, this work was carried out to study the enzymes that catalyze the hydrolysis of these compounds in plant leaves. The methods developed for assay of these enzymes in plants involve determination by steam distillation of the NH₄ ⁺–N produced by amidase or urease activity when plant materials are incubated at 37°C with buffered (0.1M THAM pH 8.0) amide solution or buffered (0.1M THAM pH 7.5) urea solution, respectively. Amidase and urease were detected in 21 diverse plants in the families of Gramineae and Leguminosae. Results showed that amidase and urease have optimum activities at buffer pH values of 8.0 and 7.5, respectively. Both amidase and urease activities were decreased significantly upon freezing or air-drying of plant samples before enzyme assay. These differences were proportional to the original activities of fresh plant materials. Studies on the effect of temperature on amidase and urease activities showed that these enzymes are inactivated at temperatures above 60 and 70°C, respectively. The energy of activation of the reaction catalyzed by amidase and urease in plants, expressed in kJ·mole^–1, ranged from 44.0 to 51.2 (avg.=47.1) and from 43.1 to 56.5 (avg.=51.2) when formamide and urea were used as substrates, respectively. The apparent K_m constants of these enzymes varied among the plant samples studied. By using the Lineweaver-Burk plot, the K_m values for amidase when formamide was used as a substrate ranged from 2.0 to 9.4 (avg.=5.8 mM) and for urease ranged from 0.4 to 1.6 (avg.=0.8 mM). The V_max values of 7 plant samples, expressed in g of NH₄ ⁺–N produced/0.1 g of plant materials/2h, ranged from 137 to 514 for amidase and from 29 to 123 for urease. The importance of these enzymes in application of amides and urea to plant leaves is discussed.     

Control of urease formation in certain aerobic bacteria

Summary During nitrogen starvation, a 20- to 250-fold increase in specific urease activity was observed in extracts of P. aeruginosa, P. fluorescens, Hydrogenomonas, M. denitrificans, M. cerificans and B. megaterium. In contrast to these species, high levels of urease were observed in P. vulgaris strains and in S. ureae under all growth conditions. No urease was detectable in strains of E. coli, S. marcescens and B. polymyxa, regardless of growth conditions.Incubated in the absence of an exogenous nitrogen source, the specific urease activity increased during a period of 10 to 20 h in P. aeruginosa, Hydrogenomonas and M. denitrificans. Phosphate starvation did not significantly effect urease formation in these strains. The increase in specific urease activity was found to be repressed by exogenous nitrogen sources, including urea. Inhibition by chloramphenicol, other inhibitors, and by the lack of oxygen or fructose, indicated that a derepressive urease formation may occur in these strains. The involvement of traces of urea possibly released from endogenous sources during starvation is discussed.     

Induction of barley leaf urease

Foliar urea application on barley plants increased leaf urease activity for 5 hours with a peak of 20-fold at 2 hours. To discern the mode of urease induction, urea with or without inhibitors and [³⁵S]methionine were incubated with leaf sections for different lengths of time. Urease was extracted, partially purified, electrophoresed, and then quantified by fluorogram. Five urease (U) isozymes were separated by PAGE. U_a and U_b might be polymers or complexes that occurred only at the peak of induced activity. U₁ and U₂ appeared at 0.5 and 0.75 hour, respectively, after urea induction, peaked at 2 hours, and persisted only in treated leaves for several additional hours indicating that they are transient inducible forms. U₃ was the constitutive form present in control and treated leaves. Induction with cordycepin or cycloheximide completely prevented urea stimulated activity and nullified the existence of isozymes U_a, U_b, U₁, and U₂. ³⁵S-U₁, which was labeled in the last hour of induction, appeared on fluorogram 1 hour after induction, peaked at 2 hours, and declined at 3 hours. Results indicated that de novo synthesis of urease is activated by the influx of urea.     

Surface Percolation for Soil Improvement by Biocementation Utilizing In Situ Enriched Indigenous Aerobic and Anaerobic Ureolytic Soil Microorganisms

The use of biocementation via microbially induced carbonate precipitation (MICP) for improving the mechanical properties of weak soils in the laboratory has gained increased attention in recent years. This study proposes an approach for applying biocementation in situ, by combining the surface percolation of nutrients and cementation solution (urea/CaCl₂) with in situ cultivation of indigenous soil urease positive microorganisms under non-sterile conditions. The enrichment of indigenous ureolytic soil bacteria was firstly tested in batch reactors. Using selective conditions (i.e., pH of 10 and urea concentrations of 0.17 M), highly active ureolytic microorganisms were enriched from four diverse soil samples under both oxygen-limited (anoxic) and oxygen-free (strictly anaerobic) conditions, providing final urease activities of more than 10 and 5 U/mL, respectively. The enrichment of indigenous ureolytic soil microorganisms was secondly tested in pure silica sand columns (300 and 1000 mm) for biocementation applications using the surface percolation approach. By applying the same selective conditions, the indigenous ureolytic soil microorganisms with high urease activity were also successfully enriched for both the fine and coarse sand columns. However, the in situ enriched urease activity was highly related to the dissolved oxygen of the percolated growth medium. The results showed that the in situ cultivated urease activity may produce non-clogging cementation over the entire 1000-mm columns, with unconfined compressive strength varying between 850–1560 kPa (for coarse sand) and 150–700 kPa (for fine sand), after 10 subsequent applications of cementation solution. The typically observed loss of ureolytic activity during the repeated application of the cementation solution was recovered by providing more growth medium under selective enrichment conditions, enabling the in situ enriched ureolytic microorganisms to increase in numbers and urease activity in such a way that continued cementation was possible.     

Morphological changes of trichophyton mentagrophytes under the influence of increased carbon dioxide tension

Summary Growth pigment production as well as macroconidia and arthrospore formation ofTrichophyton mentagrophytes are stimulated under the influence of increased carbon dioxide tension. Macroconidia with spirals and pistol forms of macroconidia are observed.The granular form ofT. mentagrophytes displays an asteroid growth (Dextrose agar of Sabouraud) with an ingrowth and elongation of the ray branches in the agar and a decrease of the air-mycelium. The culture completely resembles to those ofT. mentagrophytes varasteroides. The macroconidia appear elongated and they acquire an asteroid arrangement. The less granular strains ofT. mentagrophytes take the appearance ofT. interdigitale type.An analogy is made between these morphogenetic changes under the effect of increased CO₂ tension in vitro and the influence of the animal organism in the evolution of the growth forms of dermatophytes in vivo in the process of their parasitic adaptation.