The role of nickel in urea assimilation by algae

Nickel is required for urease synthesis by Phaeodactylum tricornutum and Tetraselmis subcordiformis and for growth on urea by Phaeodactylum. There is no requirement for nickel for urea amidolyase synthesis by Chlorella fusca var. vacuolata. Neither copper nor palladium can substitute for nickel but cobalt partially restored urease activity in Phaeodactylum. The addition of nickel to nickel-deficient cultures of Phaeodactylum or Tetraselmis resulted in a rapid increase of urease activity to 7–30 times the normal level; this increase was not inhibited by cycloheximide. It is concluded that nickel-deficient cells over-produce a non-functional urease protein and that either nickel or the functional urease enzyme participates in the regulation of the production of urease protein.Abbreviation UALase ATP; urea amidolyase     

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     

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.     

On the false positive urease activity ofYarrowia lipolytica

The ability ofYarrowia lipolytica to produce ammonia from urea was found variable on some media. The colour change of the indicator in Christensen's urea agar was not due to the urease activity of this species but was a non-specific alkalization reaction. Rapid urea broth was reliable giving no false positive results. It was found thatY. lipolytica is a urease negative yeast species.     

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     

The regulation of urease activity in Aspergillus nidulans

Aspergillus nidulans can utilize urea as a sole source of nitrogen but not as a carbon source. Urea is degraded by a urease. Mutation at any one of three genes, ureB, ureC, and ureD, may result in deficient urease activity. The ureB gene is closely linked to ureA, the structural gene for the urea transport protein. The heat lability of a ureB ^– revertant strain, intragenic complementation tests, and the linkage of ureB to ureA suggest that ureB is the urease structural gene. The ureD gene is probably involved in the synthesis or incorporation of a nickel cofactor essential for urease activity. The function of the ureC gene is not known. Urease is not induced but is subject to nitrogen regulation. The urease activities of ammonium-derepressed mutants show that the effector of nitrogen regulation is more likely to be glutamine than ammonium. When glutamine is present in the medium, urease appears to be inactivated by some means which does not involve a newly synthesized protease or a direct interaction between glutamine and urease.     

基于不同氮源培养条件的巴氏芽孢杆菌脲酶功能转录组分析

巴氏芽孢杆菌是源于土壤的革兰氏阳性菌,人们利用其高效的脲酶活性诱导产生碳酸钙的现象开发了多种应用场景.然而,巴氏芽孢杆菌的生物矿化相关代谢机制还不够明确,尤其是对在矿化作用中发挥核心作用的脲酶基因结构、表达调控机制及关联代谢等方面的研究相对较少.当前,巴氏芽孢杆菌应用研究中面临的矿化反应不可控性及不稳定性等问题都源于脲酶代谢机制的研究匮乏.因此,进一步揭示巴氏芽孢杆菌脲酶的基因信息、表达调控机制及相关代谢机理迫在眉睫.本文通过转录组测序,对比了4种培养条件下巴氏芽孢杆菌的生长情况和基因表达情况,解析了脲酶的代谢机制,结果进一步证明ATP合成与脲酶表达及尿素水解相关联,最终预测了脲酶的双操纵子结构.     

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.     

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.     

Effect of nickel deficiency in soybeans on the phytotoxicity of foliar-applied urea

The leaf-tip necrosis commonly observed after foliar fertilization of soybean [Glycine max (L.) Merr.] plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease. We recently found, however, that although addition of a urease inhibitor (phenylphosphorodiamidate) to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean leaves, it increased the leaf-tip necrosis observed after foliar fertilization. We concluded that this necrosis was due to accumulation of toxic amounts of urea rather than formation of toxic amounts of ammonia. To confirm this conclusion, we measured the urea content, urease activity, and leaf-tep necrosis of leaves of soybean plants treated with urea after growth of the plants in nutrient solutions containing different amounts of nickel (Ni), which is an essential component of urease. We found that the urease activity of these leaves decreased, and that their urea content and leaf-tip necrosis increased, with decrease in the Ni content of the nutrient solution. Besides supporting the conclusion that the leaf-tip necrosis observed after foliar fertilization of soybean with urea is due to accumulation of toxic amounts of urea in the soybean leaves, these observations indicate that Ni-deficient plants may have a lower urease activity than plants that are not deficient in Ni and may therefore be more susceptible to leaf burn when foliar-fertilized with urea.     

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.     

The effect of carbonic anhydrase,urea and urease on the calcium carbonate deposition in the shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of carbonic anhydrase (CA), urea and urease on the CaCO₃ deposition in the shell-repair membrane of the snail, Helix pomatia, was studied by injection of CA separately or in combination with urease. This treatment resulted in increased deposits of CaCO₃ and apparent crystal formation within the shell-repair membranes compared with those of the controls. The reactions to CA combined with urea were not uniform. Formation of organic crystalline structures and dendritic spherulites was observed in some of these membranes, whereas the deposition of CaCO₃ crystals was suppressed. Administration of urea alone inhibited the formation of large CaCO₃ crystals, whereas urease stimulated this process. The reaction of young snails was greater compared to adults. The membranes of young snails contained tighly packed, small CaCO₃ crystals and organic crystalline structures, which indicated increase of the calcifying centra and their successive mineralization. The results support the assumption that carbonic anhydrase and urease enhance the rate of calcium carbonate deposition and crystal formation in Helix pomatia.     

Ubiquinone and urease distribution inTaphrina andSymbiotaphrina

Identifications of ubiquinone isoprenologues are presented for isolates identified with six species ofTaphrina and for isolates of the two species ofSymbiotaphrina. All had Q-10 as the major ubiquinone system. The inclusion ofT. populina andS. buchneri, the respective type species, establishes this as the value for these genera. Both species ofSymbiotaphrina were urease positive even though, according to the literature, they are unable to utilize urea as a sole nitrogen source. The urease results for theTaphrina isolates were mixed.     

Ammonium and urea removal by Spirulina platensis

Different concentrations either of ammonium chloride or urea were used in batch and fed-batch cultivations of Spirulina platensis to evaluate the possibility of substituting nitrate by cheaper reduced nitrogen sources in wastewaters biotreatment. The maximum nitrogen concentration able to sustain the batch growth of this microalga without inhibition was 1.7 mM in both cases. Ammonium chloride was limiting for the growth at lower concentrations, whereas inhibition took place at higher levels. This inhibition effect was less marked with urea, likely because the enzymatic hydrolysis of this compound by urease controlled the ammonia transfer into the cell. Fed-batch experiments carried out by pulse-feeding either ammonium or urea proved that the use of these compounds as nitrogen sources can sustain the long term-cultivation of S. platensis, provided that the conditions for their feeding are accurately optimized.     

Rapid test for determination of urea hydrolysis

A urease test for the rapid determination of urea hydrolysis is described in which diluted urea agar concentrate was used in small amounts with dense inoculum of the test organisms. The method was evaluated and compared with Christensen's urea agar slants by using 728 clinical isolates of gram-negative bacteria. Of the 325 strains of urease-positive Proteus-Providencia-Morganella, 282 (87%) gave positive results within 5 min with the rapid test. Urease activity of 97% of these organisms became evident within 30 min. All 287 isolates which showed no urease activity on Christensen's urea agar also remained negative by this test.     

Ureolytic nitrification at low pH by Nitrosospira spec.

An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.     

Assay for soil urease activity

Summary A procedure is described that allows assay of soil urease activity. The method uses a phosphate buffer (pH 8.8) and a urea substrate concentration of 0.007 M. Incubation for 4 h at 37°C is recommended and urease activity is estimated by determining the amount of ammonium produced by urea hydrolysis in soil. The method is precise, and compares favourably with other procedures. re]19750710     

Regulation of urea uptake inPseudomonas aeruginosa

The energy-dependent urea permease was studied in two strains ofPseudomonas aeruginosa, measuring the uptake (transport and metabolism) of¹⁴C-urea. In both strains urea uptakein vivo and urease activityin vitro differed significantly with respect to kinetic parameters, temperature and pH dependence and response to metabolic inhibitors. Ammonium strongly interfered both with the expression of the urea uptake system and its activity. The inhibition of the uptake activity by ammonium was partially relieved by hydraziniumsulfate, which prevented the translocation of ammonium into the cell, and in a methylammonium/ammonium transport-defective mutant of strain DSM 50071. Furthermore, methionine-sulfoximine, which prevented the intracellular glutamine formation from ammoniumvia inhibition of glutamine synthetase, relieved the inhibition of urea uptake by ammonium. These findings suggested that urea uptake activity inP. aeruginosa is regulated by intracellular glutamine.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - GS glutamine synthetase - MSX methionine-sulfoximine     

Inactivation of allantoinase from Pseudomonas aeruginosa by a subunit of urease

Cell-free extracts prepared from Pseudomonas aeruginosa cells, cultured in a medium containing allantoin as sole source of carbon, nitrogen and energy and harvested in the stationary phase, contain an enzymicly inactive allantoinase-inhibitor complex. Pure inhibitor was isolated by dissociation of this complex followed by gelfiltration. The inhibitor had a molecular weight of about 5500 daltons. Association between inhibitor and allantoinase was demonstrated by gelfiltration and by polyacrylamide gel-electrophoresis. The inhibitor was unstable in the absence of 1 M urea and the inactivation was accompanied by aggregate formation and appearance of urease activity. The inhibitor was also isolated from cells containing urease but no allantoinase. It was concluded that the inhibitor is a subunit of urease. Inhibitors isolated from P. aeruginosa and P. acidovorans cells were active against both allantoinase from P. aeruginosa and allantoinase from P. acidovorans.