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PubMed2004

Urease activity of enterohaemorrhagic Escherichia coli depends on a specific one-base substitution in ureD.

The authors previously reported that most enterohaemorrhagic Escherichia coli (EHEC) strains do not express urease activity, despite having the urease gene. This study compared the nucleotide sequences of the urease gene clusters of a urease-activity-positive and a urease-activity-negative strain. The results showed that in the urease-activity-negative strain, ureD, a gene encoding a chaperone protein, had a single base substitution that encoded a premature stop codon resulting in a short ORF. The premature stop codon in ureD was commonly found in urease-activity-negative EHEC strains, but not in urease-activity-positive strains. Urease activity was detected after complementing the urease-activity-negative strain with ureD from the urease-activity-positive strain. Furthermore, introduction of the urease gene cluster from the urease-activity-negative strain into an amber suppressor phenotype Escherichia coli strain, DH5alpha, conferred the ability to produce the active urease. These results suggest that the lack of urease activity in most EHEC strains is due to a premature stop codon in ureD.

Amino Acid Substitution↗
PubMed1993

Bacterial ureases: structure, regulation of expression and role in pathogenesis.

The nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in either the urinary tract or the gastroduodenal region, urease acts as a virulence factor. At both sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. In addition, in the urinary tract urease activity can result in the formation of urinary calculi. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen-limiting growth conditions. For either the urea-inducible genes or the nitrogen-regulated genes transcription appears to be positively regulated. In the nitrogen-regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the LysR family of transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), a member of the AraC family of transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed.

Bacteria↗
PubMed1987

Mutational analysis of the embryo-specific urease locus of soybean.

By a non-destructive urease screen of M2 soybean (Glycine max [L.] Merr. cv. Williams) seeds, four true-breeding mutants (n4, n6, n7 and n8) were recovered which lack most (n6, n8) or all (n4, n7) embryo-specific urease activity. This trait was due to a single, recessive lesion at the Sun (seed urease-null) locus identified earlier in an exotic germplasm (PI 229324, Itachi). All sun mutants produced normal ubiquitous urease, the low abundance isozyme found in all soybean tissues examined. Tight mutants n4 and n7 accumulated no detectable embryo-specific urease protein or mRNA; n6 and n8 accumulated normal or near normal levels of urease mRNA but had seed urease protein levels approximately 5% and .05%, respectively, of the progenitor. Mutant n8 appeared to produce a low level of fully active urease (approximately 0.7% activity level, approximately 0.5% protein level) while n6 produced a higher level of an altered, nearly inactive urease (.0.09% activity level, approximately 5% protein level). Urease alterations in n6 were manifested by its increased temperature sensitivity and variation in aggregation state and pH preference. Thus, mutations in the Sun locus affected both the level and the nature of the embryo-specific urease gene products indicating that Sun encodes the embryo-specific urease. We reported earlier that the Eul locus, which controls the aggregation state of the embryo-specific urease, is on mep unit from Sun and that the Eul allele is cis to sun is not expressed (Kloth et al. 1987). That the level of urease gene product, its aggregation state and other enzyme properties can be affected by induced sun mutations, suggests that the Eul and sun alleles are at the same locus.

Genes, Plant↗
PubMed1998

Characterisation of the urease gene cluster in Bordetella bronchiseptica.

Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen. The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may potentially be involved in nickel transport. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B. bronchiseptica. We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B. bronchiseptica from phagolysosomal damage. Comparison of the urease promoter regions of B. bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B. pertussis Tohama I revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.

Amino Acid Sequence↗
PubMed2002

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.

Animals↗
PubMed2006

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium.

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Acids↗
PubMed1996

Purification and activation properties of UreD-UreF-urease apoprotein complexes.

In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.

Apoproteins↗
PubMed2005

Effects of commercial diazinon and imidacloprid on microbial urease activity in soil and sod.

Diazinon [O,O-diethyl O-2-isopropyl-6-methyl(pyrimidine-4-yl) phosphorothioate] and imidacloprid [1-(1-[6-chloro-3-pyridinyl]methyl)-N-nitro-2-imidazolidinimine] are applied to lawns for insect control simultaneously with nitrogenous fertilizers such as urea, but their potential effect on urease activity and nitrogen availability in turfgrass management has not been evaluated. Urease activity in enzyme assays, washed cell assays, and soil slurries was examined as a function of insecticide concentration. Intact cores from field sites were used to assess the effect of insecticide application on urease activity in creeping bentgrass (Agrostis palustris Huds.) and bluegrass (Poa pratensis L.) sod. Bacterial urease from Bacillus pasteurii and plant urease from jack bean [Canavalia ensiformis (L.) DC.] were unaffected by the insecticides. Both insecticides inhibited the growth of Proteus vulgaris, a urease-producing bacterium, but only diazinon significantly reduced urease activity in washed cells; neither insecticide inhibited urease activity in sonicated cells. Neither diazinon nor imidacloprid inhibited urease activity in Woolper soil (fine, mixed, mesic Typic Argiudoll) slurries, but diazinon slightly inhibited urease activity in Maury soil (fine, mixed, semiactive, mesic Typic Paleudalf) slurries. Imidacloprid had no effect on urease activity in creeping bentgrass or bluegrass sod at up to 10 times the commercial application rate. Diazinon briefly, but significantly, reduced urease activity in bluegrass sod. Co-application of imidacloprid and urea appears to be benign with respect to urease activity in soil and sod. Diazinon, in contrast, appears to have a significant, short-term, inhibitory effect on the microbial urease-producing community, but that effect depends on soil type.

Agrostis↗
PubMed1999

Characterization of metal-substituted Klebsiella aerogenes urease.

Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO2 and Ni(II); however, only approximately 15% forms active enzyme (Ni-CO2-ureaseA), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO2-ureaseB). In the absence of CO2, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO2-ureaseB species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. Xray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3-4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO2-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO2-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO2-urease. A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.

Amino Acid Substitution↗
PubMed2000

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms.

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.

Artificial Gene Fusion↗
PubMed1992

Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions.

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.

Amino Acid Sequence↗
PubMed1999

Isolation of Helicobacter pylori genes that modulate urease activity.

Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5alpha transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5alpha (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known as flhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.

ATP-Binding Cassette Transporters↗
PubMed1990

pH regulation of urease levels in Streptococcus salivarius.

Potential mechanisms for regulation of urease levels in Streptococcus salivarius were examined, including: induction by urea, nitrogen or carbon source repression, and effects of pH and CO2 (because CO2 enrichment enhanced urease detection on urea agar plates). Regulation by either pH or CO2 was confirmed by comparison of the urease accumulation pattern during anaerobic growth under CO2 with that under N2. Under CO2, there was an initial buffering plateau at pH 6.2 and a rate of Streptococcus salivarius urease accumulation three-fold that under N2, with a pH 7.6 plateau. With both gas phases there was also an increase in the rate of urease appearance coincident with the decrease in medium pH following the pH plateau. The effects of pH, CO2, and HCO3- on urease levels and on growth were separately assessed by culture in media containing 0, 25, 100 mmol/L KHCO3 buffered at different pH levels. There was an inverse relationship between the logarithm of the urease level after 24-hour growth and the pH during growth-the urease specific activity was 100-fold higher at pH 5.5, compared with pH 7.0 and above. HCO3-/CO2 (100 mmol/L) had little effect on urease levels, but was essential for growth at pH 5.5. There was no significant urease induction by urea, or repression by ammonia or glucose. There was also evidence of pH regulation of urease levels in some staphylococci, Klebsiella pneumonia, and Corynebacterium renale, but not in Actinomyces naeslundii and several other species. We conclude that the external pH is a major factor regulating urease levels in S. salivarius and possibly some other species-a mechanism equivalent to urease repression by OH-.

Actinomyces↗
PubMed2000

In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE.

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.

Bacterial Proteins↗
PubMed2001

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.

Bacterial Proteins↗
PubMed1994

In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.

The formation of active urease in Klebsiella aerogenes requires the presence of three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickel-containing enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid containing the K. aerogenes urease gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease. A molecule of native urease apoprotein is capable of binding 0, 1, 2, or 3 molecules of UreD, consistent with a trimeric structure of urease catalytic units. The UreD-urease apoprotein complexes are competent for activation by nickel, with the level of activity obtained being directly related to the number of UreD molecules bound per urease molecule. Activation of the UreD-urease complexes is rapid and accompanied by UreD dissociation. We propose that UreD is a chaperone protein which stabilizes a urease apoprotein conformation that is competent for nickel incorporation.

Apoenzymes↗
PubMed2000

Inactivation of the ptsI gene encoding enzyme I of the sugar phosphotransferase system of Streptococcus salivarius: effects on growth and urease expression.

The urease genes of Streptococcus salivarius 57.1 are tightly repressed in cells growing at neutral pH. When cells are cultivated at acidic pH values, the urease genes become derepressed and transcription is enhanced when cells are growing under carbohydrate-excess conditions. Previously, the authors proposed that the bacterial sugar:phosphotransferase system (PTS) modulated the DNA-binding activity by phosphorylation of the urease repressor when carbohydrate was limiting. The purpose of this study was to assess whether enzyme I (EI) of the PTS could be involved in modulating urease expression in response to carbohydrate availability. An EI-deficient strain (ptsI18-3) of S. salivarius 57.1 was constructed by insertional inactivation of the ptsI gene. The mutant had no measurable PTS activity and lacked EI, as assessed by Western analysis. The mutant grew as well as the wild-type strain on the non-PTS sugar lactose, and grew better than the parent when another non-PTS sugar, galactose, was the sole carbohydrate. The mutant was able to grow with glucose as the sole carbohydrate, but displayed a 24 h lag time and had a generation time some threefold longer than strain 57.1. The mean OD600 attained after 48 h by ptsI18-3 supplied with fructose was 0.16, with no additional growth observed even after 3 d. Urease expression in the wild-type and mutant strains was assessed in continuous chemostat culture. Repression of urease at neutral pH was seen in both strains under all conditions tested. Growth of wild-type cells on limiting concentrations of lactose resulted in very low levels of urease expression compared with growth on PTS sugars. In contrast, under similar conditions, urease expression in ptsI18-3 was restored to levels seen in the parent growing on PTS sugars. Growth under conditions of lactose excess resulted in further derepression of urease, but ptsI18-3 expressed about threefold higher urease activity than 57.1. The results support a role for EI in urease regulation, but also indicate that additional factors may be important in regulating urease gene expression.

Blotting, Western↗
PubMed2005

Differential regulation of urease activity in Helicobacter hepaticus and Helicobacter pylori.

Helicobacter hepaticus is a pathogen of rodents, which causes diverse enteric and hepatic inflammatory diseases and malignancies. The urease enzyme is an important colonization factor of gastric Helicobacter species like Helicobacter pylori, but little is known about the role and regulation of urease in enterohepatic Helicobacter species. Here it is reported that urease activity of H. hepaticus does not contribute to acid resistance, and that it is nickel-responsive at the post-translational level. H. hepaticus strain ATCC 51449 did not grow or survive at pH 3.0, and supplementation with urea or NiCl2 did not abrogate this acid sensitivity. Furthermore, urease enzyme activity of H. hepaticus was acid-independent, which contrasts with the acid-induced urease system of H. pylori. Nickel supplementation of Brucella medium resulted in a tenfold increase in urease activity in both H. hepaticus and H. pylori, but the maximum level of urease activity in H. hepaticus was still three- to fivefold lower when compared to H. pylori in the same conditions. The increase in urease activity of H. hepaticus was not associated with elevation of urease mRNA or protein levels. Inhibition of protein synthesis by chloramphenicol did not affect nickel-responsive induction of urease activity in H. hepaticus, and confirmed that nickel induction occurs at the post-translational level, probably by activation of preformed apo-enzyme. In conclusion, both the role of the urease enzyme and the regulation of urease activity differ between the enterohepatic pathogen H. hepaticus and the gastric pathogen H. pylori.

Gene Expression Regulation, Bacterial↗