No abstract

Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. strain TB-90, Klebsiella aerogenes, Proteus mirabilis, Helicobacter pylori, and Yersinia enterocolitica. Sequences for selected genes have been determined for at least 10 other bacterial species and the jack bean enzyme. Urease synthesis can be nitrogen regulated, urea inducible, or constitutive. The crystal structure of the K. aerogenes enzyme has been determined. When combined with chemical modification studies, biophysical and spectroscopic analyses, site-directed mutagenesis results, and kinetic inhibition experiments, the structure provides important insight into the mechanism of catalysis. Synthesis of active enzyme requires incorporation of both carbon dioxide and nickel ions into the protein. Accessory genes have been shown to be required for activation of urease apoprotein, and roles for the accessory proteins in metallocenter assembly have been proposed. Urease is central to the virulence of P. mirabilis and H. pylori. Urea hydrolysis by P. mirabilis in the urinary tract leads directly to urolithiasis (stone formation) and contributes to the development of acute pyelonephritis. The urease of H. pylori is necessary for colonization of the gastric mucosa in experimental animal models of gastritis and serves as the major antigen and diagnostic marker for gastritis and peptic ulcer disease in humans. In addition, the urease of Y. enterocolitica has been implicated as an arthritogenic factor in the development of infection-induced reactive arthritis. The significant progress in our understanding of the molecular biology of microbial ureases is reviewed.

Expression of the Escherichia coli sugar phosphate transport system, encoded by the uhpT gene, is regulated by external glucose 6-phosphate through the action of three linked regulatory genes, uhpABC. The nucleotide sequence of the uhp region cloned from Sabnonella typhimurium was determined. The deduced Uhp polypeptide sequences from the two organisms are highly related. Comparison with the corrected sequence from E. coli revealed that the four uhp genes are closely spaced, with minimal intergenic distances, and that uhpC is nearly identical in length to uhpT, both of which have substantial sequence relatedness along their entire lengths. To facilitate analysis of uhp gene function, we isolated insertions of a kanamycin resistance (Km) cassette throughout the uhp region. In-frame deletions that removed almost the entire coding region of individual or multiple uhp genes were generated by use of restriction sites at the ends of the Km cassette. The phenotypes of the Km insertions and the in-frame deletions confirmed that all three regulatory genes are required for Uhp function. Whereas the deletion of uhpA completely abolished the expression of a uhpT-lacZ reporter gene, the deletion ofuhpB or uhpC resulted in a partially elevated basal level ofexpression that was not further inducible.These results indicated that UhpB and perhaps UhpC play both positive and negative roles in the control of uhpT transcription., Translational fusions of the uhpBCT genes to topological reporter gene phoA were generated by making use of restriction sites provided by the Km cassette or with transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with models predicting that UhpC and UhpT have identical transmembrane topologies, with 10 to 12 transmembrane segments, and that UhpB has 4 to 8 amino-terminal transmembrane segments that anchor the polar carboxyl-terminal half of the protein to the cytoplasmic side of the inner membrane.

Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine-and m-fluorophenylalanine-containing toxic dipeptides. We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 p,M (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and mfluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-[3HJleucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures. We concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids.

Proteus mirabilis urease catalyzes the hydrolysis of urea, initiating the formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Purified whole-cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences was probed with a 4.2-kb BglI fragment from within the urease operon. Autoradiographs of slot blots demonstrated 4.2- and 5.8-fold increases, respectively, in urease-specific RNA upon induction with urea. Structural and accessory genes necessary for urease activity, ureD, A, B, C, E, and F, were previously cloned and sequenced (B. D. Jones and H. L. T. Mobley, J. Bacteriol. 171:6414-6422, 1989). A 1.2-kb EcoRV-BamHI restriction fragment upstream of these sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame of 882 nucleotides, designated ureR, which is transcribed in the direction opposite that of the urease structural and accessory genes and encodes a 293-amino-acid polypeptide predicted to be 33,415 Da in size. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels of [35S]methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band with an apparent molecular size of 32 kDa. Fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence contains a potential helix-turn-helix motif and an associated AraC family signature and is similar to that predicted for a number of DNA-binding proteins, including E. coli proteins that regulate acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). These data suggest that UreR governs the inducibility of P. mirabilis urease.

Expression of the Escherichia coli uhpT gene, encoding the sugar phosphate transport protein, is induced by extracellular glucose-6-phosphate and requires the function of the uhpABC regulatory genes. The UhpA and UhpB proteins are related to the response-regulator and sensor-kinase proteins of two-component regulatory systems, whereas the UhpC protein is related to UhpT and homologous transport proteins. To investigate the role of segments of the membrane-associated UhpB and UhpC regulatory proteins, a series of mutations were constructed in vitro by insertion of a 12-or 24-bp oligonucleotide linker at 44 sites within the uhpABCT locus. The effect of these mutations on regulation of a uhpT-lacZ transcriptional reporter was assayed with the mutated uhp alleles in single copy on the chromosome. All but one of the insertions in uhpA or uhpT were inactive for transcription activation or transport, respectively. In contrast, about half of the insertions in uhpB and uhpC retained Uhp expression, and insertions at four sites in uhpB and at one site in uhpC conferred high-level constitutive expression. The constitutive mutants in UhpB resulted from insertions in the nonpolar amino-terminal half of the protein, and all insertions in that half of UhpB affected Uhp expression in some manner, which suggests that the transmembrane segments of UhpB might negatively regulate the kinase activity of the carboxyl portion. The constitutive behavior of all but one of these uhpB alleles was dependent on the presence of active forms of both UhpA and UhpC, which suggests that UhpB and UhpC act jointly as a complex in the signaling process.

The transport of small peptides into the yeast Saccharomyces cerevisiae is subject to complex regulatory control. In an effort to determine the number, and to address the function, of the components involved in peptide transport and its regulation, spontaneous mutants resistant to toxic di- and tripeptides were isolated under inducing conditions. Twenty-four mutant strains were characterized in detail and fell into two phenotypic groups; one group deficient in amino acid-inducible peptide uptake, the other with a pleiotropic phenotype including a loss of peptide transport. Complementation analysis of recessive mutations in 12 of these strains defined three groups; ptr1 (nine strains), ptr2 (two strains), and ptr3 (one strain). Isolation and screening of 31 additional N-methyl-N-nitro-N-Nitrosoguanidine (MNNG)-induced, peptide transport-deficient mutants produced one ptr3 and 30 ptr2 strains: no additional complementation groups were detected. Uptake of radiolabeled dileucine was negligible in ptr1 and ptr2 strains and was reduced by 65% and 90% in the two ptr3 mutants, indicating that all strains were defective at the transport step. We conclude that the S. cerevisiae amino acid-inducible peptide transport system recognizes a broad spectrum of peptide substrates and involves at least three components. One gene, PTR3, may play an indirect or regulatory role since mutations in this gene cause a pleiotropic phenotype.

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 ,uM 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- Proteus mirabilis is not a common cause of urinary tract infection in the healthy host (31). This organism does, however, infect a high proportion of patients with complicated urinary tracts, that is, those with functional or structural abnormalities or with chronic catheterization (31, 34). In these patients, bladder and renal stone formation is a hallmark of infection with this species and is due to the expression of urease (5). The enzyme hydrolyzes urea to CO2 and NH3, often resulting in elevation of urinary pH (24). Alkalinization of the urine leads to precipitation of Ca2+, Mg2+, and other ions to form carbonate-apatite or struvite stones (5). Ureasenegative mutants, constructed by allelic exchange, are unable to form stones in transurethrally infected mice and are significantly less virulent than the urease-positive parent strains (12,13).Urease is one of only four classes of nickel metalloenzymes which also include hydrogenase, methyl coenzyme M reductase, and carbon monoxide dehydrogenase (8). These divalent cations are required for synthesis of catalytically active urease in bacteria and plants (8). It has been observed that the apourease, synthesized in the absence of nickel, is difficult to activate in vivo by the addition of nickel chloride when protein biosynthesis is inhibited (18,30). This suggests that insertion of nickel ions (Ni2+) into the metallocenter takes place primarily at the time of synthesis of the enzyme subunits, although there is some evidence to the contrary (1). Little is known, however,