Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

The effect of quinoid and phenolic compounds on urease and dehydrogenase activity and nitrification in soil

Summary 1,4-Naphthoquinone; 2-methyl-1,4-napthoquinone; 2,3-dichlorohydroquinone; 4,6-di-tert.butyl-o-benzoquinone; 4,6-di-tert.butylpyrocatechol and 4-tert.butylpyrocatechol at concentrations of 10 and 20 ppm inhibit mineralization of urea N by reducing urease activity and/or nitrification in soils. Coating of urea with these chemicals was more effective than direct application to the soil. There was no adverse effect of any of the chemicals either on germination or on the growth of wheat plants at concentrations of 20 and 50 ppm added to soil.     

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori

The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.     

Inverse nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter mustelae

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.     

Regulation of urease activity in Rhizobium meliloti

Abstract Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.     

Interaction Between Nickel and Protein Source in the Ruminant

Eighty growing steers were used to determine the effect of nickel supplementation on performance and metabolic parameters of steers fed corn silage-based diets supplemented with different crude protein sources. Crude protein sources examined included: (1) soybean meal, (2) blood meal, (3) urea, and (4) blood meal-urea (two-thirds of supplemental nitrogen from blood meal and one-third from urea). The protein sources differed in ruminal degradability, nitrogen solubility, and nickel content. Nickel was added within each protein treatment to supply either 0 or 5 ppm of supplemental nickel. The experiment was 84 d in duration and rumen fluid and blood samples were collected on days 42 and 80. Average daily gain and feed efficiency were not affected by nickel supplementation. The addition of 5 ppm supplemental nickel greatly increased rumen bacterial urease activity regardless of protein source. When samples were collected prior to feeding on day 80, nickel increased serum urea nitrogen concentrations in steers fed urea, but decreased circulating urea concentrations in animals fed blood meal or the blood meal-urea combination.Ad libitum intake of trace mineral salt was greatly reduced in steers receiving 5 ppm supplemental nickel. The present study suggests that the source of protein may influence ruminant responses to dietary nickel.     

Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.)

Nickel is considered to be an essential micronutrient in plants because of its role in the metalloenzyme urease. In order to characterize the metabolic consequences of Ni deprivation, the significance of Ni supply for growth and N metabolism of rice plants grown with either NH4NO3 or urea as sole N source was evaluated. Growth of plants receiving NH4NO3 was not affected by the Ni status, and neither were the activities of arginase and glutamine synthetase. However, urease activity was not detectable in leaves of low-Ni plants, which in conjunction with arginase action, led to the accumulation of urea in plants grown with NH4NO3. Amino acid contents and mineral nutrient status (except Ni) were not affected by the Ni treatment.Urea-grown Ni-deprived plants showed reduced growth and accumulated large amounts of urea owing to the lack of urease activity. These plants were further characterized by low amino acid contents indicating impaired usage of the N supplied. They also exhibited reduced levels of the urea precursor arginine, which is merely attributed to the overall N economy in these plant. When urea-grown plants were supplied with 0.5 mmol m^-3 Ni in the nutrient solution, the dry weight and the amino acid N contents were increased at the expense of the urea contents, indicating efficient use of urea N in Ni-supplemented plants.A critical Ni concentration in the shoot regarding dry matter production of NH4NO3-grown plants could not be deduced, while 25 g Ni kg^-1 DW is certainly inadequate for urea-grown plants. This suggests that the Ni requirement strongly depends on the N source employed.Keywords: Amino acids, ornithine cycle, Ni supply, rice, urea, urease activity.      

Effects of slow-release urea fertilizers on urease activity,microbial biomass,and nematode communities in an aquic brown soil

A field experiment was carried out at the Shenyang Experimental Station of Ecology (CAS) in order to study the effects of slow-release urea fertilizers high polymer-coated urea (SRU1), SRU1 mixed with dicyandiamide DCD (SRU2), and SRU1 mixed with calcium carbide CaC₂ (SRU3) on urease activity, microbial biomass C and N, and nematode communities in an aquic brown soil during the maize growth period. The results demonstrated that the application of slow-release urea fertilizers inhibits soil urease activity and increases the soil NH₄ ⁺-N content. Soil available N increment could promote its immobilization by microorganisms. Determination of soil microbial biomass N indicated that a combined application of coated urea and nitrification inhibitors increased the soil active N pool. The population of predators/omnivores indicated that treatment with SRU2 could provide enough soil NH₄ ⁺-N to promote maize growth and increased the food resource for the soil fauna compared with the other treatments.     

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.     

Inhibition of mineralization of urea nitrogen in soil

Summary The effects of anthraquinones and some other quinonoid and phenolic compounds on mineralization of urea N in soils were studied by estimating the influence on urease activity and nitrification. Anthraquinones did not affect the mineralization of urea N but 1,4-naphthoquinone; 2-methyl-1,4-naphthoquinone; 2–3-dichlorohydroquinone; 4,6-di-tert.butyl-o-benzoquinone; 4-tert.butylpyrocatechol and 4,6-di-tert.butylpyrocatechol inhibited urease activity and nitrification. The hydrolysis of urea (100 ppm) was not prevented by partial reduction in urease activity. The effective substances also inhibited dehydrogenase activity.     

Isolation of o-Acetylbenzene-amidinocarboxylic Acid,a New Metabolite of Gibberella saubineti

The nickel requirement and the role of nickel were investigated in a recently identified oxygen-resistant hydrogen bacterium, Xanthobacter autotrophicus strain Y38. When 0.3 μm NiSO₄ was added to the basal medium which had not been supplemented with nickel, the cell concentration of autotrophically grown strain Y38 increased by about 4-fold and the resumption of cell growth occurred in the stationary phase. These results showed the requirement of nickel for the autotrophic growth of strain Y38. Since a trace of nickel was detected in the basal medium, the role of nickel was investigated using 0.2 mm or 0.4 mm EDTA-containing media. Other trace elements, Ca, Co, Cu, Mn, Mo and Zn, could not replace nickel. Nickel was not required for the heterotrophic growth of strain Y38. Nickel seems to be related a little to urease in strain Y38. Moderate hydrogenase induction was observed in hydrogenase deficient cells of strain Y38 under 95%H₂ + 5%O₂ when 300 μm NiSO₄ was added to 0.4 mm EDTA-containing buffer but it was completely inhibited by chloramphenicol, indicating that nickel was related to the hydrogenase synthesis. A nickel dependent increase in growth rate was demonstrated equally under 40%O₂ and 10%O₂, suggesting that nickel was not directly related to the oxygen-resistance of strain Y38.     

Effects of slow-release urea fertilizers on urease activity,microbial biomass,and nematode communities in an aquic brown soil

A field experiment was carried out at the Shenyang Experimental Station of Ecology (CAS) in order to study the effects of slow-release urea fertilizers high polymer-coated urea (SRU1), SRU1 mixed with dicyandiamide DCD (SRU2), and SRU1 mixed with calcium carbide CaC₂ (SRU3) on urease activity, microbial biomass C and N, and nematode communities in an aquic brown soil during the maize growth period. The results demonstrated that the application of slow-release urea fertilizers inhibits soil urease activity and increases the soil NH₄ ⁺-N content. Soil available N increment could promote its immobilization by microorganisms. Determination of soil microbial biomass N indicated that a combined application of coated urea and nitrification inhibitors increased the soil active N pool. The population of predators/omnivores indicated that treatment with SRU2 could provide enough soil NH₄ ⁺-N to promote maize growth and increased the food resource for the soil fauna compared with the other treatments.

     

Inhibition of soil urease activity by amido derivatives of phosphoric and thiophosphoric acids

Summary A laboratory experiment was conducted to study inhibition of soil urease activity by amido derivatives of phosphoric and thiophosphoric acids. Results showed that derivatives with higher amido substitutions have greater inhibitory effect on urea hydrolysis in the soils used in our study. Triamides of phosphoric and thiophosphoric acids were found to be very effective inhibitors of soil urease. These compounds seem to have potential as fertilizer amendments for inhibiting soil urease activity and for improving the efficiency of nitrogen use from urea.     

Nickel availability and urease expression in Proteus mirabilis

Cells of Proteus mirabilis, previously grown in nutrient broth (NB), exhibited an increase in urease activity during subsequent incubation in mineral medium even when protein biosynthesis was inhibited. During growth in NB, degradation of amino acids obviously led to the formation of nickel-complexing metabolites, and nickel ions were therefore inavailable for maximal expression of enzymatically active urease; this inhibition of urcase biosynthesis was overcome by the addition of nickel to the growth medium, and also by added glucose. Experiments concerning the incorporation of radioactive nickel into urease finally indicated that the observed increase in urease activity was caused by posttranslational insertion of nickel into preformed apourease.     

砷对土壤脲酶活性影响的研究

采用模拟方法对As污染土壤脲酶特征进行了研究．结果表明,土壤中As浓度在0～200mg·kg^-1浓度范围内,反应初期脲酶活性变化无明显规律;一年后砷激活土壤脲酶活性,二者达到显著或极显著正相关．随As浓度增加,土壤脲酶Km值基本不变或略有增加,Vmax增大,从机制上揭示出As加速脲酶-尿素复合物的解离．厩肥和无肥土样脲酶对As的反应类似,只是变化幅度有所差异．     

Structural and functional role of nickel ions in urease by molecular dynamics simulation

Nickel ions play several roles in the biological processes of microorganisms and plants. Urease has a nickel-containing active site and catalyzes the hydrolysis of urea to yield ammonia and carbamate. In the present study, the role of nickel ions is examined using molecular dynamics simulations of the holo and apo forms. Nonbonded models used for the nickel ions provide good reproduction of the active-site structure as indicated in the crystallized structure. The results confirm that urease has a rigid active site in either its holo or its apo form. A new conformation of the flap is observed in apo urease. The connection between the metal center and His^α323 is proposed to be responsible for maintaining the flap conformation. The binding free energy of acetohydroxamic acid and urease is estimated using the molecular mechanics–generalized Born/surface area method. The binding free energy is primarily driven by electrostatic interactions in the presence of nickel ions. Normal mode analysis is employed to characterize the movements of the flap in apo urease.     

碳添加下黑钙土胞内、胞外脲酶活性变化及其机制

土壤脲酶作为能够催化尿素水解的最重要酶类,对草地生态系统氮素供应具有重要作用。目前探讨不同碳添加对草地土壤胞外脲酶影响的研究报道相对较多,但碳添加对土壤胞内脲酶的影响,以及胞内和胞外脲酶对碳添加的响应是否一致等尚需深入研究。本研究依托额尔古纳森林草原过渡带生态系统研究站开展的碳添加野外试验平台(以葡萄糖为碳源),选取无碳添加(C₀)、250(C₂₅₀)和500(C₅₀₀) kg C·hm^-2·a^-1处理为供试对象,探讨碳添加下黑钙土胞内、胞外脲酶活性响应及其与土壤性质的关系。结果表明: 碳添加显著提高了土壤胞内脲酶活性,增加了土壤胞内脲酶活性占总脲酶活性的比例,但对土壤胞外脲酶活性没有显著影响。土壤胞内脲酶活性与微生物生物量具有显著正相关关系,表明胞内脲酶活性增加主要是由微生物生物量增加引起的。结构方程模型(SEM)分析表明,碳添加通过影响土壤微生物生物量间接提高了土壤胞内脲酶活性。     

Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus.

Nickel is a constituent of soluble and particulate hydrogenase of Alcaligenes eutrophus. Incorporation of 63Ni2+ revealed that almost the total nickel taken up by the cells was bound to the protein. Chromatography of a crude extract on diethylaminoethyl cellulose demonstrated an association of 63Ni2+ with soluble and particulate hydrogenase, supported by further analysis like polyacrylamide gel electrophoresis. Unspecific binding of 63Ni2+ to the protein was excluded by comparison with a mutant extract free of hydrogenase protein. X-ray fluorescence analysis of the homogeneous soluble hydrogenase indicated the presence of 2 mol of nickel per mol of enzyme, whereas the amount of nickel determined by incorporation of 63Ni2+ was calculated to be approximately 1 mol/mol of enzyme. Cells grown under nickel limitation contained catalytically inactive, but serologically active, soluble and particulate hydrogenase. The immunochemical reactions were only partially identical with the enzyme from nickel-cultivated cells indicating a structural modification of the proteins in the absence of nickel. It is concluded that nickel is essential for the catalytic activity of hydrogenase and not involved as a regulatory component in the synthesis of this enzyme.