Nickel Supplementation Research Articles

Effect of nickel supplementation on growth performance and blood metabolites of growing Muzaffarnagari lambs

Effect of nickel supplementation on growth performance and blood metabolites of growing Muzaffarnagari lambs

Iron and Nickel Supplementation Exerts a Significant Positive Effect on the Hydrogen and Methane Production from Organic Solid Waste in a Two-Stage Digestion

Two-stage anaerobic digestion and trace metals (TM) supplementation are promising techniques to improve biogas production. Fe2+ and Ni2+ can improve process stability since they are part of the cofactors of enzymes and microorganisms’ growth. This work attempted to evaluate the effect of Fe2+ and Ni2+ addition on H2-rich biogas production from organic solid waste and the CH4-rich biogas production from the acidogenic effluents (AEs) enriched with TM. The TM concentrations that enhanced the hydrogen yield in the batch were 0.25 mg/L of Ni2+ and 334 mg/L of Fe2+. These concentrations were evaluated in a two-stage system. The substrate for the batch tests and fermentative reactor (first stage) was OSW. The AE generated in the first stage was the substrate to produce CH4-rich biogas in the second stage. In the first stage, the productivity achieved was 1823 ± 160 mL H2/L/day. However, TM supplementation decreased productivity by 65% since the VS removal increased. Megasphaera genus predominated in the first stage. Regarding the methanogenic reactor, the undiluted AE without TM caused the fast decay of the process. Nevertheless, the reactor operated stably after using AE enriched with TM as a substrate, and CH4 yields increased by 42%. The highest productivity achieved in the second stage was 1278 ± 42 mL CH4/L/day, operating with an organic loading rate of 2.8 gVS/L/day. The genera Proteiniphilum, Thermovirga, DMER64, Anaerovorax, and Syntrophomonas predominated in the second stage. In conclusion, AE enriched with TM can be used to recover the stability of anaerobic digesters, increasing methane production.

Effects of nickel supplementation on nutrient utilization, mineral balance, haematology and antioxidant status of crossbred dairy calves

BackgroundNickel has been identified as an important newer trace mineral playing essential role in animals however, its precise mechanism of action in the body is yet to be determined. Interaction of Ni with other essential minerals is suggested in reports limited to lab animals and needs to be explored further in large animals. AimThis study was conducted to study the influence of Ni supplementation at different levels, on minerals and health status of crossbred dairy calves. MethodTwenty-four Karan Fries crossbred (Tharparkar × Holstein Friesian) male dairy calves were selected based on body weight (137.09 ± 5.68) and age (10.78 ± 0.61) and were divided into 4 treatment groups (n = 6) given basal diet supplemented with 0 (Ni0), 5 (Ni5), 7.5 (Ni7.5) and 10 (Ni10) ppm Ni/kg of DM. Nickel was supplemented in the form of nickel sulphate hexahydrate (NiSO4.6 H2O) solution. To ensure the intake of the required amount of nickel by each animal, the calculated quantity of solution was mixed with 250 g concentrate mixture and offered individually to the calves. The calves were fed total mixed ration (TMR) consisting of green fodder, wheat straw and concentrate mixture in the ratio of 40:20:40 and the nutritional requirements were met according to NRC (2001) guidelines. Growth performance was recorded at fortnightly interval whereas, plasma minerals, haematology, antioxidant and immunity parameters were studied at monthly interval during the 150-day experimental period. Nutrient utilization and mineral balances were estimated with the help of a metabolism trial conducted at the end of feeding trial. ResultsSupplementation of Ni exhibited no influence on dry matter intake (DMI), body weight, average daily gain (ADG) and nutrient digestibility of dairy calves. However, the absorption and balance of minerals such as Ni, Fe, Cu, Zn and their respective plasma concentration increased (P < 0.05) with Ni supplementation and highest values were observed in calves fed 10 mg Ni/kg DM. The red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (HCT) and activity of superoxide dismutase (SOD) and catalase antioxidant enzymes showed highest increase (P < 0.05) in calves supplemented with Ni at level of 10 mg/kg DM as compared to other treatment groups. However, white blood cell (WBC) count, glutathione peroxidase (GPx), total antioxidant status (TAS), total immunoglobulins and IgG plasma concentration remained unaltered with addition of different levels of Ni in the diet of calves. ConclusionsThe supplementation of Ni at level of 10 mg/kg DM shows a positive effect on status of trace minerals such as Fe, Cu, Zn and improves the physiological conditions and health status of crossbred dairy calves indicated by improved haematology and antioxidant parameters.

Influence of supplementary nickel on feed intake, nutrient utilization and growth performance in Murrah buffalo calves

Eighteen male Murrah buffalo calves of about similar age (8.7 months) and body weight (125 kg) were selected from Livestock Research Centre, ICAR- National Dairy Research Institute, Karnal, Haryana, India and divided into 3 groups of 6animals using randomised block design to investigate the effect of different levels of nickel (Ni) supplementation on feed intake, nutrient utilization, growth performance and rumen fermentation parameter. All the animals were fed to meet their nutrient requirements ICAR (2013), however, the animals in groups T1, T2and T3 were supplemented with 0, 5 and 10 ppm of Ni, respectively. Daily DM intake and fortnightly body weights were recorded in the morning before offering feed and water. Rumen liquor samples were collected at 0, 60 and 120 days of the experiment to analyze different rumen fermentation variables. Different levels of Ni in the diet did not influence nutrient intake, digestibility of nutrients(DM, OM, CP, EE, NDF and ADF), nitrogen balance, growth rate and feed conversion ratio. The values of various rumen fermentation parameters like pH, total volatile fatty acids, ammonia-N and TCA precipitable-N were similar in the three groups. The urease activity in rumen liquor was the highest(P&lt;0.05) in group T3. The propionate level increased (P&lt;0.05)while that of butyrate decreased in group T3 as compared to groups T1 and T2 showing no significant effect on acetate concentration. Hence, supplementation of Ni upto 10 ppm in the ration did not affect nutrient utiliisation and growth performance in male Murrah buffalo calves, however, urease activity and proportion of propionate increased to a greater extent in calves supplemented with 10 ppm Ni.

Effect of nickel supplementation on growth, haematology, biomarkers of energy and protein metabolism of heifers fed urea based diet

Effect of nickel supplementation on growth, haematology, biomarkers of energy and protein metabolism of heifers fed urea based diet

Urease production using corn steep liquor as a low-cost nutrient source by Sporosarcina pasteurii: biocementation and process optimization via artificial intelligence approaches.

To commercialize the biocementation through microbial induced carbonate precipitation (MICP), the current study aimed at replacing the costly standard nutrient medium with corn steep liquor (CSL), an inexpensive bio-industrial by-product, on the production of urease enzyme by Sporosarcina pasteurii (PTC 1845). Multiple linear regression (MLR) in linear and quadratic forms, adaptive neuro-fuzzy inference system (ANFIS), and genetic programming (GP) were used for modeling of process based on the experimental data for improving the urease activity (UA). In these models, CSL concentration, urea concentration, nickel supplementation, and incubation time as independent variables and UA as target function were considered. The results of modeling showed that the GP model had the best performance to predict the extent of urease, compared to other ones. The GP model had higher R2 as well as lower RSME in comparison with the models derived from ANFIS and MLR. Under the optimum conditions optimized by GP method, the maximum UA value of 3.6 Mm min-1 was also obtained for 5%v/v CSL concentration, 4.5 g L-1 urea concentration, 0 μM nickel supplementation, and 60 h incubation time. A good agreement between the outputs of GP model for the optimal UA and experimental result was obtained. Finally, a series of laboratory experiments were undertaken to evaluate the influence of biological cementation on the strengthening behavior of treated soil. The maximum shear stress improvement between bio-treated and untreated samples was 292% under normal stress of 55.5 kN as a result of an increase in interparticle cohesion parameters.

Effects of foam nickel supplementation on anaerobic digestion: Direct interspecies electron transfer

Stimulating direct interspecies electron transfer with conductive materials is a promising strategy to overcome the limitation of electron transfer efficiency in syntrophic methanogenesis of industrial wastewater. This paper assessed the impact of conductive foam nickel (FN) supplementation on syntrophic methanogenesis and found that addition of 2.45 g/L FN in anaerobic digestion increased the maximum methane production rate by 27.4 % (on day 3) while decreasing the peak production time by 33 % as compared to the control with no FN. Cumulative methane production from day 2 to 6 was 14.5 % higher with addition of 2.45 g/L FN than in the control. Levels of FN in excess of 2.45 g/L did not show benefits. Cyclic voltammetry results indicated that the biofilm formed on the FN could generate electrons. The dominant bacterial genera in suspended sludge were Dechlorobacter and Rikenellaceae DMER64, whereas that in the FN biofilm was Clostridium sensu stricto 11. The dominant archaea Methanosaeta in the FN biofilm was enriched by 14.1 % as compared to the control.

Effect of Nickel Supplementation on Liver and Kidney Function Test and Protein Metabolism in Growing Cattle

This study was conducted to assess the nickel (Ni) content of commonly available feedstuffs and their supplemental effect on biomarkers of liver and kidney function and protein metabolism in growing cattle. Eighteen growing Hariana heifers were randomly assigned to three groups for 90 days and managed on similar feeding regimen except that these three groups were supplemented with 0.0 (Ni0.0), 1.5 (Ni1.5), and 3.0 (Ni3.0) of Ni/kg DMI. Cereal grain by-products, cakes and meals, green fodders, molasses, and compounded concentrate were high in Ni content. Cereal grains, straw, and stovers were moderate to low in Ni content. Dietary supplementation of 3.0 mg of Ni/kg DMI showed linear increase (P < 0.01) in average daily gain. No effects of treatments were observed on haemoglobin concentration, haematocrit value, aspartate aminotransferase level, alanine aminotransferase level, and alkaline phosphatase level. Circulating levels of bilirubin (P < 0.05) and creatinine (P < 0.01) showed dose-dependent increase with supplemental Ni. Heifers receiving diet supplemented with Ni showed higher (P < 0.001) plasma urease activity, plasma levels of total protein, albumin, globulin, and plasma urea nitrogen as compared to non-supplemented heifers. Ni supplementation showed a trend of linear increase (P < 0.001) in plasma Ni concentrations, and Ni level was observed highest in Ni3.0 group. Mean plasma iron (Fe) concentration showed no effect of Ni supplementation. The results of the present study indicate that Ni supplementation seems to improve performance in growing cattle by modulating urease activity and protein metabolism.

Effects of Nickel Supplementation on Antioxidant Status, Immune Characteristics, and Energy and Lipid Metabolism in Growing Cattle.

Nickel (Ni) has not been elucidated as an essential mineral in dairy animals, though in plants and lower organisms, its role in activation of urease enzyme is well known. This study was conducted to evaluate the effect of Ni supplementation on intake, growth performance, urease activity, antioxidant and immune status, and energy and lipid metabolism in growing cattle. Eighteen growing Hariana heifers were randomly allocated into three groups on body weight (125 ± 3.0kg) and age basis (10 ± 2.0months). Feeding regimen was similar in all the groups except that treatment groups were supplemented with 0.0 (Ni0.0), 1.5 (Ni1.5), and 3.0 (Ni3.0) mg of Ni/kg dry matter (DM) in three respective groups. DM intake (DMI), average daily gain (ADG), feed efficiency, plasma urease activity, biomarkers of antioxidant and immune status, energy and lipid metabolism, and plasma Ni levels were observed during the 90-day experimental period. There was linear increase (p< 0.05) in mean DMI and ADG without affecting feed efficiency was observed in 3.0mg of Ni/kg DM supplemented heifers. Dietary Ni supplementation showed linear increase (p < 0.05) in mean plasma urease activity. No effects of (p > 0.05) of Ni supplementation were observed on superoxide dismutase (SOD) and catalase (CAT) activity and plasma lipid peroxide (LPO) concentration; whereas, mean plasma total antioxidant status (TAS) showed linear decrease (p < 0.001) in Ni-supplemented groups. Adding Ni up to 3.0mg of Ni/kg DM did not exert (p > 0.05) any effect on plasma total immunoglobulin and immunoglobulin G (IgG) concentrations. Mean plasma cortisol level showed negative association with supplemental Ni levels and concentration was found lowest (p < 0.05) in 3.0mg of Ni/kg DM-added group. Dietary Ni supplementation did not affect mean plasma concentrations of glucose, cholesterol, triglyceride, and non-esterified fatty acids (NEFA). There was a linear increase (p< 0.001) in plasma Ni concentrations as the Ni concentrations increased in the diet. The results of present study indicated that dietary supplementation of 3.0mg of Ni/kg DM improved performance of growing cattle by increasing urease activity.

Fluctuations in Foodborne Nickel as an Outcome of Industrial Processing

Foodborne nickel content was determined in six strains of oat in a year-on-year analysis. Results are depicted by relative standard deviation (RSD) values of 17.94 and 31.41%. Wider range of differential is featured in processed foods, with RSD spanning 10–65%. While most studies are confined to analyses in output data only, in this article we dissect the mechanisms that underlie nickel migration in the progress of industrial processing. After-effects attributed to manufacturing are tracked with graphite furnace atomic absorption spectrometer throughout the production cycle. As it starts with raw harvest, nickel content often exceeds 600 ng/g. Most is discarded when middlings are cleaned into grains and after bran is separated. Although there is little discrepancy between checkpoint stages of flour and bread (), it is the intermediate phases where nickel content fluctuations are most pronounced. Procedure involving numerous backslopping steps favors nickel pool enrichment, while thermal processing was found to entail its reduction. Practical Applications Unintentional nickel supplementation during food processing exerts influence on the dietary properties of produced commodities. This is especially significant taken relatively large number of hypersensitive consumers, whose best interest is to limit daily nickel intake. We examined bread production cycles in their thoroughness and gathered data on ever changing nickel content with an inclusion of sequential intermediate phases that are rarely of analytical interest. These phases give an insight into subtle changes reflected in chemical composition of bakery products. If account is taken of presented knowledge, it will allow more aware manufacturing practice and, in consequence, higher quality and nutritional safety of produced foodstuffs.

Nickel accumulation in leaves, floral organs and rewards varies by serpentine soil affinity.

Serpentine soils are edaphically stressful environments that host many endemic plant species. In particular, serpentine soils are high in several heavy metals (e.g. nickel, cobalt and chromium) and these high heavy metal concentrations are thought, in part, to lead to varying levels of plant adaptation and soil affinities (i.e. endemic vs. non-endemic plant species). It is unclear, however, whether serpentine endemics vs. non-endemics differ with respect to heavy metal uptake into either vegetative or reproductive organs. Here, we use nickel as a model to determine whether plant heavy metal uptake varies with the level of endemism in several non-hyperaccumulating species. Under controlled greenhouse conditions, we grew seven plant species from the Brassicaceae family that vary in their degrees of affinity to serpentine soil from low (indifferent) to medium (indicator) and high (endemic) in soil that was nickel supplemented or not. We quantified nickel concentrations in leaves, pistils, anthers, pollen and nectar. While nickel concentrations did not vary across organs or affinities when grown in control soils, under conditions of nickel supplementation endemic species had the lowest tissue concentrations of nickel, particularly when considering leaves and pistils, compared with indifferent/indicator species. Species indifferent to serpentines incorporated higher concentrations of nickel into reproductive organs relative to leaves, but this was not the case for indicator species and endemics where nickel concentration was similar in these organs. Our findings suggest that endemic species possess the ability to limit nickel uptake into above-ground tissues, particularly in reproductive organs where it may interfere with survival and reproduction. Indifferent species accumulated significantly more nickel into reproductive organs compared with leaves, which may limit their reproductive potential relative to endemic species when growing on serpentine soils. Additional work determining the fitness consequences of these differences will further our understanding of edaphic endemism.

Identification and Structure of a Novel Archaeal HypB for [NiFe] Hydrogenase Maturation

HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a Kd value of 84nM. ADP binds more tightly than does ATP, with a Kd value of 15nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea.

Identification and structure of a novel archaeal HypB for [NiFe] hydrogenase maturation

Abstract HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a Kd value of 84 nM. ADP binds more tightly than does ATP, with a Kd value of 15 nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea.

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Abstract Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H 2 is rapidly consumed by the uptake hydrogenase. This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL − (deficient in the uptake hydrogenase), hoxH − (deficient in the bidirectional hydrogenase) and hupL − /hoxH − (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N 2 and/or CO 2 addition), light intensity (54 and 152 μEm −2 s −1 ), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium. In every assay, the hupL − and hupL − /hoxH − mutants stood out over wild-type cells and the hoxH − mutant. Nevertheless, the hupL − mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 μEm −2 s −1 in the light period, with 1 μM of NiCl 2 supplementation in the culture medium, and under a propane atmosphere. In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO 2 addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO 2 plus 1% of N 2 did not affect positively hydrogen production rate.

Nickel supplementation effect on the growth, urease activity and urea and nitrate concentrations in lettuce supplied with different nitrogen sources

Effect of nickel (Ni) in the nutrient solution on yield, N metabolism, and nitrate content of leafy vegetables is poorly understood. The aim of this nutrient solution culture experiment was to investigate the effects of Ni supplementation on the nitrogen (N) metabolism and growth of lettuce ( Lactuca sativa L. cv. Baker) with either urea or nitrate as the N source. Nickel supplement at 0.04 μM reduced urea toxicity to the urea-fed plants. Addition of Ni to the nutrient solution significantly increased the leaves and root growth of the urea-fed lettuce plants while it increased the growth of lettuce plants fed with nitrate only at N level of 20 mM. At N level of 20 mM, the leaves fresh weight of the urea-fed plants promoted by Ni supplement was comparable with the nitrate-fed plants untreated with Ni. Nickel supplementation increased the leaf total N concentrations in the urea-fed plants, although the nitrate-fed plants accumulated greater N in their leaves compared with urea treated plants at without Ni treatment. Nickel addition decreased the concentrations of leaf urea-N in the urea-fed plants and NO 3-N in the nitrate-fed plants. Nickel addition enhanced urease activity in the leaves of urea-fed plants. The results indicated Ni supplementation enhances the growth of the urea-fed lettuce plants while it has role in decreasing leaf nitrate concentration and thus, improving the health quality of the nitrate-fed plants.

The CRR1 Nutritional Copper Sensor in Chlamydomonas Contains Two Distinct Metal-Responsive Domains

Copper response regulator 1 (CRR1), an SBP-domain transcription factor, is a global regulator of nutritional copper signaling in Chlamydomonas reinhardtii and activates genes necessary during periods of copper deficiency. We localized Chlamydomonas CRR1 to the nucleus in mustard (Sinapis alba) seedlings, a location consistent with its function as a transcription factor. The Zn binding SBP domain of CRR1 binds copper ions in vitro. Cu(I) can replace Zn(II), but the Cu(II) form is unstable. The DNA binding activity is inhibited in vitro by Cu(II) or Hg(II) ions, which also prevent activation of transcription in vivo, but not by Co(II) or Ni(II), which have no effect in vivo. Copper inhibition of DNA binding is reduced by mutation of a conserved His residue. These results implicate the SBP domain in copper sensing. Deletion of a C-terminal metallothionein-like Cys-rich domain impacted neither nutritional copper signaling nor the effect of mercuric supplementation, but rendered CRR1 insensitive to hypoxia and to nickel supplementation, which normally activate the copper deficiency regulon in wild-type cells. Strains carrying the crr1-ΔCys allele upregulate ZRT genes and hyperaccumulate Zn(II), suggesting that the effect of nickel ions may be revealing a role for the C-terminal domain of CRR1 in zinc homeostasis in Chlamydomonas.

Enhancement of anaerobic hydrogen production by iron and nickel

The effects of iron and nickel on hydrogen (H 2) production were investigated in a glucose-fed anaerobic Continuous Flow Stirred Tank Reactor (ACSTR). Both iron and nickel improved the reactor performance and H 2 production was enhanced by 71% with the sole iron or nickel supplementation. In all cases, H 2 production yield was increased by lowering both ethanol and total metabolites production and increasing butyrate production. Furthermore, iron and nickel slightly increased biomass production while glucose degradation decreased with the supplementation of nickel. Dynamic changes in bacterial composition as analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) revealed that hydrogen was produced mainly by Clostridium butyricum strains and that nickel addition decreased the microbial diversity.

Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity.

Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX(5)DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.

Host-Dependent Expression of Rhizobium leguminosarum bv. viciae Hydrogenase Is Controlled at Transcriptional and Post-Transcriptional Levels in Legume Nodules

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

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.