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     

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.     

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.     

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.     

Urease activity inTrichophyton mentagrophytes

Urease activity was detected in the dermatophyteTrichophyton mentagrophytes cells at early exponential phase of growth. Specific activity of urease decreased with culture age. At exogenous urea concentrations above 2 mm formation of urease was inhibited. The pH optimum lay at 7–7.5, the K_m being 14 mm. No urease activity could be detected in cell-free culture fluid ofT. mentagrophytes. No endoor exocellular urease activity could be detected in aT. rubrum strain grown with or without urea.     

Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 × 10⁻⁵, then multiplied 19% more rapidly than low urease cells. This frequency is about 10³ greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

Prototheca zopfii (12 strains) is able to use glucose, fructose, propanol, glycerol, and acetate as sources of carbon for growth. One of the strains is biochemically (utilization also of galactose and mannose), and two strains are morphologically slightly different.Two strains can be identified as P. wikerhamii. They exhibit good growth with glucose, fructose, galactose, trehalose, propanol, glycerol, acetate, and glutamate as sources of carbon. P. spec. 263-2 grows only with glucose and acatate. P. zopfii and P. wickerhamii are able to use urea, glycine, and glutamate as sources of nitrogen. P. spec. 263-2, on the other hand, cannot utilize these organic nitrogen compounds for growth.Four strains of Chlorella protothecoides are able to use glucose, fructose, galactose, and acetate as sources of carbon for growth in the dark. Three of them utilize also mannose, trehalose, and glutamate. Two strains can grow with glycerol, and one is able to use lactose. — Urea and glycine can serve as sources of nitrogen for the four strains of C. protothecoides. Glutamate supports growth of three strains, and one strain is able to use nicotinamide.     

Effect of Availability of Nitrogen Compounds on Community Structure of Aquatic Bacteria in Model Systems

To test if the quality and concentration of dissolved nitrogen (N) species could be a selective force in shaping bacterioplankton community structure, competition for various N compounds among five heterotrophic marine bacteria (Pseudomonas strains B, B25, and AX; Bacillus strain A6; Erythrobacter strain F19) was examined. Two of the five strains (AX and B25) were capable of utilizing urea for growth. The five strains were inoculated into dilute (1/1,000 strength) ZoBell medium enriched with various N sources (free amino acids, casein, ammonium, nitrate, or urea). Regardless of the added N source, the communities were either dominated by strain B (at 50 μM N) or strain AX (at 250 μM N). Without any addition of N, strain F19 dominated. If F19 was not included in the community, strain B25 dominated. Despite these differences in community structure, consumption of the added N compounds was surprisingly similar and no advantages of urea for the urea-utilizing bacterium B25 were obvious. To examine if urea could be of selective advantage to the urea-degrading strains B25 and AX, communities with and without B25 were amended with urea N. As expected, strain B25 became dominant when present, but without this strain the non-urea-utilizing strain B outcompeted the urea-utilizing strain AX. Possibly, strain B benefited from N released during catabolism of urea by strain AX. Changes in community composition did not result in major changes in the nitrogen dynamics. The results indicate that dissolved N species can be a selective force in shaping microbial communities. Relative to nutrient generalists, nutrient specialists may either have competitive advantages or stimulate growth of other species by synergetic interactions. Results from the model communities suggest that there may be a large degree of unpredictability in the making of microbial communities, whereas major ecosystem functions such as N cycling appear relatively stable.     

Urea uptake and urease activity in Corynebacterium glutamicum

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10^–7 cm s^–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K _m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (K _m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min^–1 (mg dry wt.)^–1]. Received: 11 August 1997 / Accepted: 2 December 1997     

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     

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     

Role of nitrogen sources and metal ions in urease synthesis byMicrococcus varians

Urease (urea amidohydrolase E.C.3.5.1.5) synthesis inMicrococcus varians U-9 was not affected by nitrogen source (peptone or glutamic acid) or its concentration: but depended on the ratio of peptone and urea in culture medium. WhenM. varians grew in culture medium with peptone at or above 0.48g/l and NH₄Cl as an additional nitrogen source, two maxima of urease synthesis occurred; one in the exponential growth phase and the second in the stationary growth phase. Though this bacterium could not utilize either urea or ammonia as the sole nitrogen source, urea caused only one maximum of urease synthesis to occur and shifted the maximum into late exponential phase, suggesting that urea acts as a regulatory factor in urease synthesis. Synthesis of urease was not induced either by urea or by nitrogen starvation and was not repressed by ammonia or by excess of complex nitrogen source. NI²⁺ (up to 0.1 mM) stimulated urease synthesis but decreased bacterial growth, while Co²⁺ only affected bacterial growth and at 0.1 mM Inhibited the growth.

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Résumé La synthèse d'uréase (urée-amidohydrolase E.C.3.5.1.5) chezMicrococcus varians n'est pas affectée par la source d'azote (peptone ou acide glutamique), ni par leurs concentrations, mais dépend du rapport de la peptone à l'urée dans le milieu de culture. QuandM. varians croît dans un milieu de culture contenant la peptone à la concentration égale ou supérieure à 0.48 g/l et le NH₄Cl comme source additionnelle d'azote, on observe deux maximum de synthèse d'uréase: la première dans la phase exponentielle de croissance, et la seconde dans la phase stationnaire de croissance. Blen que cette bactérie ne puisse utilliser ni l'urée ni l'ammoniac comme seule source d'azote, l'urée provoque un seul des maximum de synthèse d'uréase et déplace ce maximum vers la partie ultime de la phase exponentielle, suggérant que l'urée agit comme facteur de régulation dans la synthèse d'uréase. La synthèse d'uréase n'est induite ni par l'urée ni par la starvation d'azote et n'est réprimée ni par l'ammoniac ni par un excès de la source d'azote complexe. Le Ni²⁺ (jusqu'à 0.1 mM) stimule la synthèse d'uréase, mais diminue la croissance bactérienne, tandis que le Co²⁺ n'affecte que la croissance bactérienne et à 0.1 mM inhibe la croissance.

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This work was carried out at the Faculty of Food Technology and Biotechnology, University of Zagreb. Department of Biochemical Engineering, 41000 Zagreb, Pierottijeva 6/IV, Croatia, Yugoslavia.     

Antibacterial,antifungal and cytotoxic properties of novel N-substituted sulfonamides from 4-hydroxycoumarin

A new series of 4-({[2, 4-dioxo-2H-chromen-3 (4H)-ylidene] methyl} amino) sulfonamides have been obtained by the condensation reaction of 4-hydroxycoumarin with various sulfonamides (sulfanilamide, sulfaguanidine, p-aminomethylsufanilamide, p-aminoethylsufanilamide, sulfathiazole, sulfamethoxazole, sulfamethazine and 4-[(2-amino-4-pyrimidinyl) amino] benzenesulfonamide) in the presence of an excess of ethylorthoformate. These compounds were screened for their in-vitro antibacterial activity against four Gram-negative (E. coli, S. flexneri, P. aeruginosa and S. typhi) and two Gram-positive (B. subtilis and S. aureus) bacterial strains and for in-vitro antifungal activity against T. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glaberata. Results revealed that a significant antibacterial activity was observed by compounds (4) and (5), (6) and (8) against two Gram-negative, (P. aeruginosa and S. typhi) and two Gram-positive (B. subtilis and S. aureus) species, respectively. Of these (4) was found to be the most active. Similarly, for antifungal activity compounds (3) and (8) showed significant activity against M. canis and, (6) and (8) against F. solani. The brine shrimp bioassay was also carried out to study their in-vitro cytotoxic properties and only two compounds, (4) and (8) possessing LD₅₀ = 2.9072 × 10^{? 4} and 3.2844 × 10^{? 4} M, respectively, displayed potent cytotoxic activity against Artemia salina     

Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.     

Diagnostic Criteria for Differentiation of Pseudomonads Pathogenic for Man

To determine the most useful diagnostic characters for the differentiation of pseudomonads pathogenic for man, including Pseudomonas maltophilia (Alcaligenes bookeri), P. stutzeri (Bacillus denitrificans), P. pseudomallei (Malleomyces pseudomallei), and apyocyanogenic strains of P. aeruginosa, a comparative examination was made of 58 strains of these bacilli for their morphological and biochemical characteristics. It was concluded that the criteria for differentiating these bacteria are type of flagellation, growth on S S Agar, fluorescein production, oxidation of carbohydrates in the medium of Hugh and Leifson, nitrogen gas production, gluconate oxidation, gelatinase activity, urease activity, lysine decarboxylase activity, arginine dihydrolase activity, oxidase reaction, sensitivity to polymyxin, requirement for methionine as a growth factor, and assimilation of organic compounds as the sole source of carbon and energy.     

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.     

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.