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.     

Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli.

Ureaplasma urealyticum is a pathogenic ureolytic mollicute which colonizes the urogenital tracts of humans. A genetic polymorphism between the two biotypes of U. urealyticum at the level of the urease genes was found. The urease gene cluster from a biotype 1 representative of U. urealyticum (serotype I) was cloned and sequenced. Seven genes were found, with ureA, ureB, and ureC encoding the structural subunits and ureE, ureF, ureG, and a truncated ureI) gene encoding accessory proteins. Urease expression was not obtained when the plasmid containing these genes was incorporated into an opal suppressor strain of Escherichia coli, although this enzymatic activity was found in the same E. coli strain transformed with pC6b, a plasmid with previously cloned urease genes from the U. urealyticum T960 strain of biotype 2 (serotype 8). Although there are 12 TGA triplets encoding tryptophan within urease genes, the level of expression obtained was comparable to the levels reported for other bacterial genes expressed in E. coli. Nested deletion experiments allowed us to demonstrate that ureD is necessary for urease activity whereas another open reading frame located downstream is not. The promoter for ureA and possibly other urease genes was identified for both serotypes.     

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.     

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations.     

细菌脲酶蛋白复合物及其活化机制

脲酶能够催化尿素分解生成氨,在农业和医学领域中具有重要的意义。细菌脲酶蛋白包括结构蛋白(UreA、UreB和UreC)和辅助蛋白(UreD/UreH、UreE、UreF和UreG),它们在脲酶活化过程中各自具有独特的作用,结构蛋白形成脲酶活性中心,而辅助蛋白主要负责镍离子的传递。文中综述了细菌脲酶蛋白复合物的结构和功能,以及各蛋白之间如何相互作用完成其活化过程,以期为脲酶活性调控研究及脲酶抑制剂开发等提供理论指导。     

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

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.     

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.     

Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation.

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.     

Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange.

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.     

The urease structural gene ureA in Rhizobium meliloti is preceded by an open reading frame necessary for urease activity

Abstract An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis . Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.     

Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.     

Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.     

Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease.

In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.     

Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a UreD-UreF-UreG complex bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. This study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein-protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in copurification of UreD and urease apoprotein, whereas UreG bound to only a subset of the species. Notably, weakened interaction with UreG correlated with the low-activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD-UreF-UreG-urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the noncognate metal Zn.