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The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided.

Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.

A diaziridinylspermine analogue, 1,12-diaziridinyl-4,9-diazadodecane (NSC-667005), was synthesized as a bisalkylating agent with a polyamine backbone. DNA cross-linking was detected in the reaction of linearized pBR322 DNA with 1,12-diaziridinyl-4,9-diazadodecane at concentrations comparable with that required for cross-linking by two nitrogen mustard drugs, mechlorethamine and melphalan. A significant increase in life span of female CD2F1 mice bearing L1210 murine leukemia was observed after intravenous administration of 1,12-diaziridinyl-4,9-diazadodecane in doses of less than 2.7 mg/kg, given on days 1, 5, and 9 of treatment.

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.

Helicobacter pylori has been demonstrated as an etiologic agent of human gastritis and peptic ulcer formation. However, there is no straightforward basis to distinguish different isolates. We used the polymerase chain reaction (PCR) to amplify the urease structural subunit genes, ureA and ureB, which, when digested with appropriate restriction endonucleases, allow the differentiation of patterns on agarose gels. PCR amplification was possible with DNA rapidly extracted from H. pylori by alkaline lysis and phenol-chloroform. The 2.4-kb PCR products amplified from 22 clinical isolates and subjected to HaeII restriction endonuclease digestion produced 10 distinct patterns on agarose gels, with two patterns being shared between five and six strains. PCR amplification of the urease genes may enable the differentiation of closely related H. pylori strains by restriction digest analysis of PCR-amplified ureA and ureB genes.