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1.  Comparative and Functional Genomic Analysis of Prokaryotic Nickel and Cobalt Uptake Transporters: Evidence for a Novel Group of ATP-Binding Cassette Transporters†  
Journal of Bacteriology  2006;188(1):317-327.
The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B12 riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B12 biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.
doi:10.1128/JB.188.1.317-327.2006
PMCID: PMC1317602  PMID: 16352848
2.  Molybdoproteomes and evolution of molybdenum utilization 
Journal of molecular biology  2008;379(4):881-899.
Summary
The trace element molybdenum (Mo) is utilized in many life forms, and it is a key component of several enzymes involved in nitrogen, sulfur, and carbon metabolism. With the exception of nitrogenase, Mo is bound in proteins to a pterin, thus forming the molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. Although a number of molybdoenzymes are well characterized structurally and functionally, evolutionary analyses of Mo utilization are limited. Here, we carried out comparative genomic and phylogenetic analyses to examine the occurrence and evolution of Mo utilization in bacteria, archaea and eukaryotes at the level of (i) Mo transport and Moco utilization trait, and (ii) Mo-dependent enzymes. Our results revealed that most prokaryotes and all higher eukaryotes utilize Mo whereas many unicellular eukaryotes including parasites and most yeasts lost the ability to use this metal. In addition, eukaryotes have fewer molybdoenzyme families than prokaryotes. Dimethylsulfoxide reductase (DMSOR) and sulfite oxidase (SO) families were the most widespread molybdoenzymes in prokaryotes and eukaryotes, respectively. A distant group of the ModABC transport system, was predicted in the hyperthermophilic archaeon Pyrobaculum. ModE-type regulation of Mo uptake occurred in less than 30% of Moco-utilizing organisms. A link between Mo and selenocysteine utilization in prokaryotes was also identified wherein the selenocysteine trait was largely a subset of the Mo trait, presumably due to formate dehydrogenase, a Mo- and selenium-containing protein. Finally, analysis of environmental conditions and organisms that do or do not depend on Mo revealed that host-associated organisms and organisms with low G+C content tend to reduce their Mo utilization. Overall, our data provide new insights into Mo utilization and show its wide occurrence, yet limited use of this metal in individual organisms in all three domains of life.
doi:10.1016/j.jmb.2008.03.051
PMCID: PMC2670968  PMID: 18485362
molybdenum; molybdopterin; molybdoenzyme; comparative genomics; evolution
3.  A Computational Framework for Proteome-Wide Pursuit and Prediction of Metalloproteins using ICP-MS and MS/MS Data 
BMC Bioinformatics  2011;12:64.
Background
Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins.
Results
We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions.
Conclusions
We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.
doi:10.1186/1471-2105-12-64
PMCID: PMC3058030  PMID: 21356119
4.  Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation 
PLoS Genetics  2009;5(9):e1000652.
Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.
Author Summary
Effects of mutations can change under different genetic backgrounds or environmental factors, also known as epistasis and genotype-by-environment interactions (G×E), respectively. Though epistasis and G×E are traditionally treated as distinct phenomena, our study of a beneficial mutation highlights their commonality. This mutation resulted from insertion of the same transposable element upstream of a novel cobalt transport system in 30 of 32 independent populations during evolution in metal-limited media. The resulting increased cobalt uptake provided a selective benefit that depended upon two environmental factors: cobalt limitation and growth substrates whose metabolism requires a particular vitamin B12 (which contains cobalt) -dependent biochemical pathway. Furthermore, this mutation exhibited epistatic and G×E interactions with other cellular processes in a generic way, such that its selective advantage increased as cells were able to grow faster. This growth-rate dependence accords with a simple model: the slowest of multiple physiological processes needed for growth exerts the greatest control over an organism's growth rate. It suggests that as growth results from the performance of the entire physiological system, genes or environmental factors that affect distinct physiological processes may thus interact through their convergent effects on growth phenotypes.
doi:10.1371/journal.pgen.1000652
PMCID: PMC2732905  PMID: 19763169
5.  An ABC Transporter and a TonB Ortholog Contribute to Helicobacter mustelae Nickel and Cobalt Acquisition▿ †  
Infection and Immunity  2010;78(10):4261-4267.
The genomes of Helicobacter species colonizing the mammalian gastric mucosa (like Helicobacter pylori) contain a large number of genes annotated as iron acquisition genes but only few nickel acquisition genes, which contrasts with the central position of nickel in the urease-mediated acid resistance of these gastric pathogens. In this study we have investigated the predicted iron and nickel acquisition systems of the ferret pathogen Helicobacter mustelae. The expression of the outer membrane protein-encoding frpB2 gene was iron and Fur repressed, whereas the expression of the ABC transporter genes fecD and ceuE was iron and Fur independent. The inactivation of the two tonB genes showed that TonB1 is required for heme utilization, whereas the absence of TonB2 only marginally affected iron-dependent growth but led to reduced cellular nickel content and urease activity. The inactivation of the fecD and ceuE ABC transporter genes did not affect iron levels but resulted in significantly reduced urease activity and cellular nickel content. Surprisingly, the inactivation of the nixA nickel transporter gene affected cellular nickel content and urease activity only when combined with the inactivation of other nickel acquisition genes, like fecD or ceuE. The FecDE ABC transporter is not specific for nickel, since an fecD mutant also showed reduced cellular cobalt levels and increased cobalt resistance. We conclude that the H. mustelae fecDE and ceuE genes encode an ABC transporter involved in nickel and cobalt acquisition, which works independently of the nickel transporter NixA, while TonB2 is required primarily for nickel acquisition, with TonB1 being required for heme utilization.
doi:10.1128/IAI.00365-10
PMCID: PMC2950367  PMID: 20643857
6.  Roles of His-Rich Hpn and Hpn-Like Proteins in Helicobacter pylori Nickel Physiology▿  
Journal of Bacteriology  2007;189(11):4120-4126.
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.
doi:10.1128/JB.01245-06
PMCID: PMC1913394  PMID: 17384182
7.  Is the Nickel-Dependent Urease Complex of Cryptococcus the Pathogen’s Achilles’ Heel? 
mBio  2013;4(4):e00408-13.
ABSTRACT
The nitrogen-scavenging enzyme urease has been coopted in a variety of pathogenic organisms as a virulence factor, most notoriously to neutralize stomach acid and establish infection by the gastric pathogen Helicobacter pylori. The opportunistic fungal pathogen Cryptococcus neoformans also utilizes urease as a virulence factor, only in this case to invade the central nervous system (CNS) via the blood-brain barrier and cause life-threatening meningoencephalitis. A recent study [A. Singh, R. Panting, A. Varma, T. Saijo, K. Waldron, A. Jong, P. Ngamskulrungroj, Y. Chan, J. Rutherford, K. Kwon-Chung, mBio 4(3):e00220-13] genetically and biochemically characterizes the accessory proteins required for successful activation of the urease protein complex, including the essential nickel cofactor. The accessory proteins Ure4, Ure6, and Ure7 are all essential for urease function. Ure7 appears to combine the roles of two bacterial accessory proteins: it incorporates both the GTPase activity and nickel chaperone properties of UreE, a bacterial protein whose homolog is missing in the fungi. An accompanying nickel transporter, Nic1, is responsible for most, but not all, nickel uptake into the fungal cell. Mutants of the core urease protein Ure1, accessory protein Ure7, and transporter Nic1 are all attenuated for invasion of the CNS of mice, and urease activity may directly affect integrity of the tight junction of the endothelial cells of the blood-brain barrier, the network of proteins that limits paracellular permeability. This work highlights the potential of urease, its accessory proteins, and nickel transport as potential chemotherapeutic targets.
doi:10.1128/mBio.00408-13
PMCID: PMC3697809  PMID: 23800398
8.  Spectrometric and Voltammetric Analysis of Urease – Nickel Nanoelectrode as an Electrochemical Sensor 
Sensors (Basel, Switzerland)  2007;7(7):1238-1255.
Urease is the enzyme catalyzing the hydrolysis of urea into carbon dioxide and ammonia. This enzyme is substrate-specific, which means that the enzyme catalyzes the hydrolysis of urea only. This feature is a basic diagnostic criterion used in the determination of many bacteria species. Most of the methods utilized for detection of urease are based on analysis of its enzyme activity – the hydrolysis of urea. The aim of this work was to detect urease indirectly by spectrometric method and directly by voltammetric methods. As spectrometric method we used is called indophenol assay. The sensitivity of detection itself is not sufficient to analyse the samples without pre-concentration steps. Therefore we utilized adsorptive transfer stripping technique coupled with differential pulse voltammetry to detect urease. The influence of accumulation time, pH of supporting electrolyte and concentration of urease on the enzyme peak height was investigated. Under the optimized experimental conditions (0.2 M acetate buffer pH 4.6 and accumulation time of 120 s) the detection limit of urease evaluated as 3 S/N was 200 ng/ml. The activity of urease enzyme depends on the presence of nickel. Thus the influence of nickel(II) ions on electrochemical response of the enzyme was studied. Based on the results obtained the interaction of nickel(II) ions and urease can be determined using electrochemical methods. Therefore we prepared Ni nanoelectrodes to measure urease. The Ni nanoelectrodes was analysed after the template dissolution by scanning electron microscopy. The results shown vertically aligned Ni nanopillars almost covered the electrode surface, whereas the defect places are minor and insignificant in comparison with total electrode surface. We were able to not only detect urease itself but also to distinguish its native and denatured form.
PMCID: PMC3923183
urease; electrochemical methods; nanotechnology; nanotube; nickel electrode; hanging mercury drop electrode; spectrometry
9.  Characterization of Bacillus anthracis arginase: effects of pH, temperature, and cell viability on metal preference 
BMC Biochemistry  2008;9:15.
Background
Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea. While previously characterized arginases have an alkaline pH optimum and require activation with manganese, arginase from Helicobacter pylori is optimally active with cobalt at pH 6. The arginase from Bacillus anthracis is not well characterized; therefore, this arginase was investigated by a variety of strategies and the enzyme was purified.
Results
The rocF gene from B. anthracis was cloned and expressed in E. coli and compared with E. coli expressing H. pylori rocF. In the native organisms B. anthracis arginase was up to 1,000 times more active than H. pylori arginase and displayed remarkable activity in the absence of exogenous metals, although manganese, cobalt, and nickel all improved activity. Optimal B. anthracis arginase activity occurred with nickel at an alkaline pH. Either B. anthracis arginase expressed in E. coli or purified B. anthracis RocF showed similar findings. The B. anthracis arginase expressed in E. coli shifted its metal preference from Ni > Co > Mn when assayed at pH 6 to Ni > Mn > Co at pH 9. Using a viable cell arginase assay, B. anthracis arginase increased dramatically when the cells were grown with manganese, even at final concentrations of <1 μM, whereas B. anthracis grown with cobalt or nickel (≥500 μM) showed no such increase, suggesting existence of a high affinity and specificity manganese transporter.
Conclusion
Unlike other eubacterial arginases, B. anthracis arginase displays unusual metal promiscuity. The unique properties of B. anthracis arginase may allow utilization of a specific metal, depending on the in vivo niches occupied by this organism.
doi:10.1186/1471-2091-9-15
PMCID: PMC2423185  PMID: 18522738
10.  Factors Required for Activation of Urease as a Virulence Determinant in Cryptococcus neoformans 
mBio  2013;4(3):e00220-13.
ABSTRACT
Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria.
IMPORTANCE
Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans
doi:10.1128/mBio.00220-13
PMCID: PMC3663189  PMID: 23653445
11.  Genome-Wide Transcriptional Response of the Archaeon Thermococcus gammatolerans to Cadmium 
PLoS ONE  2012;7(7):e41935.
Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO4) and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM) and a toxic (1 mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.
doi:10.1371/journal.pone.0041935
PMCID: PMC3407056  PMID: 22848664
12.  Interplay of metal ions and urease 
Summary
Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.
doi:10.1039/b903311d
PMCID: PMC2745169  PMID: 20046957
13.  Genes Encoding Specific Nickel Transport Systems Flank the Chromosomal Urease Locus of Pathogenic Yersiniae 
Journal of Bacteriology  2002;184(20):5706-5713.
The transition metal nickel is an essential cofactor for a number of bacterial enzymes, one of which is urease. Prior to its incorporation into metalloenzyme active sites, nickel must be imported into the cell. Here, we report identification of two loci corresponding to nickel-specific transport systems in the gram-negative, ureolytic bacterium Yersinia pseudotuberculosis. The loci are located on each side of the chromosomal urease gene cluster ureABCEFGD and have the same orientation as the latter. The yntABCDE locus upstream of the ure genes encodes five predicted products with sequence homology to ATP-binding cassette nickel permeases present in several gram-negative bacteria. The ureH gene, located downstream of ure, encodes a single-component carrier which displays homology to polypeptides of the nickel-cobalt transporter family. Transporters with homology to these two classes are also present (again in proximity to the urease locus) in the other two pathogenic yersiniae, Y. pestis and Y. enterocolitica. An Escherichia coli nikA insertion mutant recovered nickel uptake ability following heterologous complementation with either the ynt or the ureH plasmid-borne gene of Y. pseudotuberculosis, demonstrating that each carrier is necessary and sufficient for nickel transport. Deletion of ynt in Y. pseudotuberculosis almost completely abolished bacterial urease activity, whereas deletion of ureH had no effect. Nevertheless, rates of nickel transport were significantly altered in both ynt and ureH mutants. Furthermore, the ynt ureH double mutant was totally devoid of nickel uptake ability, thus indicating that Ynt and UreH constitute the only routes for nickel entry. Both Ynt and UreH show selectivity for Ni2+ ions. This is the first reported identification of genes coding for both kinds of nickel-specific permeases situated adjacent to the urease gene cluster in the genome of a microorganism.
doi:10.1128/JB.184.20.5706-5713.2002
PMCID: PMC139606  PMID: 12270829
14.  Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease 
PLoS Biology  2013;11(10):e1001678.
Structural and biochemical study of urease accessory protein complex provides mechanistic insights into the delivery of nickel to metalloenzyme urease, an enzyme enabling the survival of Helicobacter pylori in the human stomach.
Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.
Author Summary
Catalytic activities of many important enzymes depend upon metal cofactors. Ensuring each enzyme acquires the proper type of metal cofactor is essential to life. One such example is urease, which is a nickel containing metalloenzyme catalyzing the hydrolysis of urea to ammonia. The survival of Helicobacter pylori, a stomach ulcer–causing pathogen, in the human stomach depends on the ammonia released to neutralize gastric acid. In this study, we revealed the detail mechanism of how urease accessory proteins UreF, UreH, and UreG cooperate to couple GTP hydrolysis to deliver nickel to urease. UreF/UreH complex interacts with two molecules of GTPase UreG and assembles a metal binding site located at the interface between two UreG molecules. Nickel can induce GTP-dependent dimerization of UreG. This nickel-carrying UreG dimer together with UreF, UreH, and urease assemble into a protein complex. Upon stimulation of UreG GTPase activity by bicarbonate, UreG hydrolyses GTP and releases nickel into urease. Other nickel-delivering NTPases share similar properties with UreG; therefore, the nickel delivery mechanism described here is likely universally shared among these proteins.
doi:10.1371/journal.pbio.1001678
PMCID: PMC3792862  PMID: 24115911
15.  Molecular evolution of urea amidolyase and urea carboxylase in fungi 
Background
Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms.
Results
Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes) had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota). It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina). Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina.
Conclusions
We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal gene transfer event from beta-, gamma-, or related species of proteobacteria. The event took place before the divergence of the subphyla Pezizomycotina and Saccharomycotina but after the divergence of the subphylum Taphrinomycotina. Urea carboxylase genes currently found in fungi and other limited organisms were also likely derived from another ancestral gene in bacteria. Our study presented another important example showing plastic and opportunistic genome evolution in bacteria and fungi and their evolutionary interplay.
doi:10.1186/1471-2148-11-80
PMCID: PMC3073912  PMID: 21447149
16.  Microbial ureases: significance, regulation, and molecular characterization. 
Microbiological Reviews  1989;53(1):85-108.
Microbial ureases hydrolyze urea to ammonia and carbon dioxide. Urease activity of an infectious microorganism can contribute to the development of urinary stones, pyelonephritis, gastric ulceration, and other diseases. In contrast to these harmful effects, urease activity of ruminal and gastrointestinal microorganisms can benefit both the microbe and host by recycling (thereby conserving) urea nitrogen. Microbial ureases also play an important role in utilization of environmental nitrogenous compounds and urea-based fertilizers. Urease is a high-molecular-weight, multimeric, nickel-containing enzyme. Its cytoplasmic location requires that urea enter the cell for utilization, and in some species energy-dependent urea uptake systems have been detected. Eucaryotic microorganisms possess a homopolymeric urease, analogous to the well-studied plant enzyme composed of six identical subunits. Gram-positive bacteria may also possess homopolymeric ureases, but the evidence for this is not conclusive. In contrast, ureases from gram-negative bacteria studied thus far clearly possess three distinct subunits with Mrs of 65,000 to 73,000 (alpha), 10,000 to 12,000 (beta), and 8,000 to 10,000 (gamma). Tightly bound nickel is present in all ureases and appears to participate in catalysis. Urease genes have been cloned from several species, and nickel-containing recombinant ureases have been characterized. Three structural genes are transcribed on a single messenger ribonucleic acid and translated in the order gamma, beta, and then alpha. In addition to these genes, several other peptides are encoded in the urease operon of some species. The roles for these other genes are not firmly established, but may involve regulation, urea transport, nickel transport, or nickel processing.
PMCID: PMC372718  PMID: 2651866
17.  Phytoplankton responses to atmospheric metal deposition in the coastal and open-ocean Sargasso Sea 
This study investigated the impact of atmospheric metal deposition on natural phytoplankton communities at open-ocean and coastal sites in the Sargasso Sea during the spring bloom. Locally collected aerosols with different metal contents were added to natural phytoplankton assemblages from each site, and changes in nitrate, dissolved metal concentration, and phytoplankton abundance and carbon content were monitored. Addition of aerosol doubled the concentrations of cadmium (Cd), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), and nickel (Ni) in the incubation water. Over the 3-day experiments, greater drawdown of dissolved metals occurred in the open ocean water, whereas little metal drawdown occurred in the coastal water. Two populations of picoeukaryotic algae and Synechococcus grew in response to aerosol additions in both experiments. Particulate organic carbon increased and was most sensitive to changes in picoeukaryote abundance. Phytoplankton community composition differed depending on the chemistry of the aerosol added. Enrichment with aerosol that had higher metal content led to a 10-fold increase in Synechococcus abundance in the oceanic experiment but not in the coastal experiment. Enrichment of aerosol-derived Co, Mn, and Ni were particularly enhanced in the oceanic experiment, suggesting the Synechococcus population may have been fertilized by these aerosol metals. Cu-binding ligand concentrations were in excess of dissolved Cu in both experiments, and increased with aerosol additions. Bioavailable free hydrated Cu2+ concentrations were below toxicity thresholds throughout both experiments. These experiments show (1) atmospheric deposition contributes biologically important metals to seawater, (2) these metals are consumed over time scales commensurate with cell growth, and (3) growth responses can differ between distinct Synechococcus or eukaryotic algal populations despite their relatively close geographic proximity and taxonomic similarity.
doi:10.3389/fmicb.2012.00359
PMCID: PMC3470407  PMID: 23181057
atmospheric metal deposition; colimitation; copper toxicity; incubation; nutrient addition experiment; picoeukaryote; Prochlorococcus; Synechococcus
18.  Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of Helicobacter pylori 
Journal of Bacteriology  2002;184(5):1438-1443.
Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79—both part of the characteristic sequence motif—and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.
doi:10.1128/JB.184.5.1438-1443.2002
PMCID: PMC134868  PMID: 11844775
19.  Interaction of Carcinogenic Metals with Tissue and Body Fluids 
British Journal of Cancer  1972;26(4):279-291.
In addition to cobalt, metallic nickel, cadmium and other metals (e.g. zinc and copper) dissolve when incubated with horse serum at 37°. The dissolving property of copper in serum resembles that of cobalt, its solubility being increased greatly in the presence of oxygen, whereas the solubilities of cadmium, zinc and most preparations of nickel are the same aerobically and anaerobically. In all of these “metal-sera” the cations are bound, although in different proportions, both by proteins and by small diffusible molecules.
Although Co2+ ions and cobalt-serum cause limited catalytic oxidation of fresh serum, most of the oxygen uptake by suspensions of metallic cobalt in serum, or by more simple model systems, is due to absorption of oxygen by the metal powder; the consequences of this are discussed.
Metallic cobalt, cadmium and nickel dissolve readily when incubated with sterile homogenates of rat muscle (and other tissues), the dissolved cations being bound predominantly by small, diffusible molecules, rather than by the protein components. Binding by small molecules, in preference to proteins, also occurs when the metals dissolve in vivo. In both the in vivo and in vitro systems, the metallic ions are not bound by a specific cation carrier, but are distributed amongst a number of components. These components have greater affinities for the dissolved metals than serum proteins and seem likely to be the normal cation carriers in vivo. As in serum, solubility in muscle homogenates is not a specific property of the carcinogenic metals as other, non-carcinogenic metals also dissolve. The specificity of the former metals, therefore, is attributed to the subsequent effects of the dissolved cations after intracellular incorporation.
PMCID: PMC2008656  PMID: 5071190
20.  Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II 
Journal of Bacteriology  2002;184(13):3569-3577.
HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.
doi:10.1128/JB.184.13.3569-3577.2002
PMCID: PMC135128  PMID: 12057951
21.  MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements▿ † 
Journal of Bacteriology  2009;191(19):5976-5987.
We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of ∼40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel- and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.
doi:10.1128/JB.00465-09
PMCID: PMC2747888  PMID: 19648243
22.  Evolution of SET-domain protein families in the unicellular and multicellular Ascomycota fungi 
Background
The evolution of multicellularity is accompanied by the occurrence of differentiated tissues, of organismal developmental programs, and of mechanisms keeping the balance between proliferation and differentiation. Initially, the SET-domain proteins were associated exclusively with regulation of developmental genes in metazoa. However, finding of SET-domain genes in the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that SET-domain proteins regulate a much broader variety of biological programs. Intuitively, it is expected that the numbers, types, and biochemical specificity of SET-domain proteins of multicellular versus unicellular forms would reflect the differences in their biology. However, comparisons across the unicellular and multicellular domains of life are complicated by the lack of knowledge of the ancestral SET-domain genes. Even within the crown group, different biological systems might use the epigenetic 'code' differently, adapting it to organism-specific needs. Simplifying the model, we undertook a systematic phylogenetic analysis of one monophyletic fungal group (Ascomycetes) containing unicellular yeasts, Saccharomycotina (hemiascomycetes), and a filamentous fungal group, Pezizomycotina (euascomycetes).
Results
Systematic analysis of the SET-domain genes across an entire eukaryotic phylum has outlined clear distinctions in the SET-domain gene collections in the unicellular and in the multicellular (filamentous) relatives; diversification of SET-domain gene families has increased further with the expansion and elaboration of multicellularity in animal and plant systems. We found several ascomycota-specific SET-domain gene groups; each was unique to either Saccharomycotina or Pezizomycotina fungi. Our analysis revealed that the numbers and types of SET-domain genes in the Saccharomycotina did not reflect the habitats, pathogenicity, mechanisms of sexuality, or the ability to undergo morphogenic transformations. However, novel genes have appeared for functions associated with the transition to multicellularity. Descendents of most of the SET-domain gene families found in the filamentous fungi could be traced in the genomes of extant animals and plants, albeit as more complex structural forms.
Conclusion
SET-domain genes found in the filamentous species but absent from the unicellular sister group reflect two alternative evolutionary events: deletion from the yeast genomes or appearance of novel structures in filamentous fungal groups. There were no Ascomycota-specific SET-domain gene families (i.e., absent from animal and plant genomes); however, plants and animals share SET-domain gene subfamilies that do not exist in the fungi. Phylogenetic and gene-structure analyses defined several animal and plant SET-domain genes as sister groups while those of fungal origin were basal to them. Plants and animals also share SET-domain subfamilies that do not exist in fungi.
doi:10.1186/1471-2148-8-190
PMCID: PMC2474616  PMID: 18593478
23.  Comparative assessment of performance and genome dependence among phylogenetic profiling methods 
BMC Bioinformatics  2006;7:420.
Background
The rapidly increasing speed with which genome sequence data can be generated will be accompanied by an exponential increase in the number of sequenced eukaryotes. With the increasing number of sequenced eukaryotic genomes comes a need for bioinformatic techniques to aid in functional annotation. Ideally, genome context based techniques such as proximity, fusion, and phylogenetic profiling, which have been so successful in prokaryotes, could be utilized in eukaryotes. Here we explore the application of phylogenetic profiling, a method that exploits the evolutionary co-occurrence of genes in the assignment of functional linkages, to eukaryotic genomes.
Results
In order to evaluate the performance of phylogenetic profiling in eukaryotes, we assessed the relative performance of commonly used profile construction techniques and genome compositions in predicting functional linkages in both prokaryotic and eukaryotic organisms. When predicting linkages in E. coli with a prokaryotic profile, the use of continuous values constructed from transformed BLAST bit-scores performed better than profiles composed of discretized E-values; the use of discretized E-values resulted in more accurate linkages when using S. cerevisiae as the query organism. Extending this analysis by incorporating several eukaryotic genomes in profiles containing a majority of prokaryotes resulted in similar overall accuracy, but with a surprising reduction in pathway diversity among the most significant linkages. Furthermore, the application of phylogenetic profiling using profiles composed of only eukaryotes resulted in the loss of the strong correlation between common KEGG pathway membership and profile similarity score. Profile construction methods, orthology definitions, ontology and domain complexity were explored as possible sources of the poor performance of eukaryotic profiles, but with no improvement in results.
Conclusion
Given the current set of completely sequenced eukaryotic organisms, phylogenetic profiling using profiles generated from any of the commonly used techniques was found to yield extremely poor results. These findings imply genome-specific requirements for constructing functionally relevant phylogenetic profiles, and suggest that differences in the evolutionary history between different kingdoms might generally limit the usefulness of phylogenetic profiling in eukaryotes.
doi:10.1186/1471-2105-7-420
PMCID: PMC1592128  PMID: 17005048
24.  Comparative Genomic Analyses of Copper Transporters and Cuproproteomes Reveal Evolutionary Dynamics of Copper Utilization and Its Link to Oxygen 
PLoS ONE  2008;3(1):e1378.
Copper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. We found that copper use is widespread among prokaryotes, but also identified several phyla that lack cuproproteins. This is in contrast to the use of other trace elements, such as selenium, which shows more scattered and reduced usage, yet larger selenoproteomes. Copper transporters had different patterns of occurrence than cuproproteins, suggesting that the pathways of copper utilization and copper detoxification are independent of each other. We present evidence that organisms living in oxygen-rich environments utilize copper, whereas the majority of anaerobic organisms do not. In addition, among copper users, cuproproteomes of aerobic organisms were larger than those of anaerobic organisms. Prokaryotic cuproproteomes were small and dominated by a single protein, cytochrome c oxidase. The data are consistent with the idea that proteins evolved to utilize copper following the oxygenation of the Earth.
doi:10.1371/journal.pone.0001378
PMCID: PMC2147054  PMID: 18167539
25.  Identification and Characterization of the Nickel Uptake System for Urease Biogenesis in Streptococcus salivarius 57.I 
Journal of Bacteriology  2003;185(23):6773-6779.
Ureases are multisubunit enzymes requiring Ni2+ for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni2+ uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3′ to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni2+ uptake in S. salivarius, regions 3′ to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate 63Ni2+ during growth. Supplementation of the growth medium with NiCl2 at concentrations as low as 2.5 μM partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni2+ was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni2+-specific ATP-binding cassette transporter.
doi:10.1128/JB.185.23.6773-6779.2003
PMCID: PMC262724  PMID: 14617641

Results 1-25 (770098)