Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1992 Jul;60(7):2657–2666. doi: 10.1128/iai.60.7.2657-2666.1992

Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB.

L T Hu 1, P A Foxall 1, R Russell 1, H L Mobley 1
PMCID: PMC257217  PMID: 1612735

Abstract

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.

Full text

PDF
2657

Images in this article

2658Image
on p.2658
2660Image
on p.2660
2660Image
on p.2660
2661Image
on p.2661
2662Image
on p.2662

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  2. Bell G. D., Weil J., Harrison G., Morden A., Jones P. H., Gant P. W., Trowell J. E., Yoong A. K., Daneshmend T. K., Logan R. F. 14C-urea breath analysis, a non-invasive test for Campylobacter pylori in the stomach. Lancet. 1987 Jun 13;1(8546):1367–1368. doi: 10.1016/s0140-6736(87)90664-7. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clayton C. L., Pallen M. J., Kleanthous H., Wren B. W., Tabaqchali S. Nucleotide sequence of two genes from Helicobacter pylori encoding for urease subunits. Nucleic Acids Res. 1990 Jan 25;18(2):362–362. doi: 10.1093/nar/18.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clayton C. L., Wren B. W., Mullany P., Topping A., Tabaqchali S. Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12. Infect Immun. 1989 Feb;57(2):623–629. doi: 10.1128/iai.57.2.623-629.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cussac V., Ferrero R. L., Labigne A. Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. J Bacteriol. 1992 Apr;174(8):2466–2473. doi: 10.1128/jb.174.8.2466-2473.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dent J. C., McNulty C. A., Uff J. S., Gear M. W., Wilkinson S. P. Campylobacter pylori urease: a new serological test. Lancet. 1988 Apr 30;1(8592):1002–1002. doi: 10.1016/s0140-6736(88)91827-2. [DOI] [PubMed] [Google Scholar]
  8. Dunn B. E., Campbell G. P., Perez-Perez G. I., Blaser M. J. Purification and characterization of urease from Helicobacter pylori. J Biol Chem. 1990 Jun 5;265(16):9464–9469. [PubMed] [Google Scholar]
  9. Eaton K. A., Brooks C. L., Morgan D. R., Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991 Jul;59(7):2470–2475. doi: 10.1128/iai.59.7.2470-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evans D. J., Jr, Evans D. G., Kirkpatrick S. S., Graham D. Y. Characterization of the Helicobacter pylori urease and purification of its subunits. Microb Pathog. 1991 Jan;10(1):15–26. doi: 10.1016/0882-4010(91)90062-f. [DOI] [PubMed] [Google Scholar]
  11. Farmer J. J., 3rd, Hickman F. W., Brenner D. J., Schreiber M., Rickenbach D. G. Unusual Enterobacteriaceae. "Proteus rettgeri" that "change" into Providencia stuartii. J Clin Microbiol. 1977 Oct;6(4):373–378. doi: 10.1128/jcm.6.4.373-378.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foxall P. A., Hu L. T., Mobley H. L. Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates. J Clin Microbiol. 1992 Mar;30(3):739–741. doi: 10.1128/jcm.30.3.739-741.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gherardini F. C., Hobbs M. M., Stamm L. V., Bassford P. J., Jr Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum. J Bacteriol. 1990 Jun;172(6):2996–3002. doi: 10.1128/jb.172.6.2996-3002.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graham D. Y., Klein P. D., Evans D. J., Jr, Evans D. G., Alpert L. C., Opekun A. R., Boutton T. W. Campylobacter pylori detected noninvasively by the 13C-urea breath test. Lancet. 1987 May 23;1(8543):1174–1177. doi: 10.1016/s0140-6736(87)92145-3. [DOI] [PubMed] [Google Scholar]
  15. Hamilton-Miller J. M., Gargan R. A. Rapid screening for urease inhibitors. Invest Urol. 1979 Mar;16(5):327–328. [PubMed] [Google Scholar]
  16. Hausinger R. P. Nickel utilization by microorganisms. Microbiol Rev. 1987 Mar;51(1):22–42. doi: 10.1128/mr.51.1.22-42.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hawtin P. R., Delves H. T., Newell D. G. The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy. FEMS Microbiol Lett. 1991 Jan 1;61(1):51–54. doi: 10.1016/0378-1097(91)90012-y. [DOI] [PubMed] [Google Scholar]
  18. Hopkins R. J., Russell R. G., O'Donnoghue J. M., Wasserman S. S., Lefkowitz A., Morris J. G., Jr Seroprevalence of Helicobacter pylori in Seventh-Day Adventists and other groups in Maryland. Lack of association with diet. Arch Intern Med. 1990 Nov;150(11):2347–2348. [PubMed] [Google Scholar]
  19. Hu L. T., Mobley H. L. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect Immun. 1990 Apr;58(4):992–998. doi: 10.1128/iai.58.4.992-998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hu L. T., Nicholson E. B., Jones B. D., Lynch M. J., Mobley H. L. Morganella morganii urease: purification, characterization, and isolation of gene sequences. J Bacteriol. 1990 Jun;172(6):3073–3080. doi: 10.1128/jb.172.6.3073-3080.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones B. D., Mobley H. L. Genetic and biochemical diversity of ureases of Proteus, Providencia, and Morganella species isolated from urinary tract infection. Infect Immun. 1987 Sep;55(9):2198–2203. doi: 10.1128/iai.55.9.2198-2203.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones B. D., Mobley H. L. Proteus mirabilis urease: genetic organization, regulation, and expression of structural genes. J Bacteriol. 1988 Aug;170(8):3342–3349. doi: 10.1128/jb.170.8.3342-3349.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones B. D., Mobley H. L. Proteus mirabilis urease: nucleotide sequence determination and comparison with jack bean urease. J Bacteriol. 1989 Dec;171(12):6414–6422. doi: 10.1128/jb.171.12.6414-6422.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Labigne A., Cussac V., Courcoux P. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J Bacteriol. 1991 Mar;173(6):1920–1931. doi: 10.1128/jb.173.6.1920-1931.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lee M. H., Mulrooney S. B., Hausinger R. P. Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme. J Bacteriol. 1990 Aug;172(8):4427–4431. doi: 10.1128/jb.172.8.4427-4431.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mobley H. L., Cortesia M. J., Rosenthal L. E., Jones B. D. Characterization of urease from Campylobacter pylori. J Clin Microbiol. 1988 May;26(5):831–836. doi: 10.1128/jcm.26.5.831-836.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mobley H. L., Hausinger R. P. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev. 1989 Mar;53(1):85–108. doi: 10.1128/mr.53.1.85-108.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mobley H. L., Jones B. D., Jerse A. E. Cloning of urease gene sequences from Providencia stuartii. Infect Immun. 1986 Oct;54(1):161–169. doi: 10.1128/iai.54.1.161-169.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mulrooney S. B., Hausinger R. P. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J Bacteriol. 1990 Oct;172(10):5837–5843. doi: 10.1128/jb.172.10.5837-5843.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Newell D. G., Stacey A. Antigens for the serodiagnosis of Campylobacter pylori infections. Gastroenterol Clin Biol. 1989;13(1 Pt 1):37B–41B. [PubMed] [Google Scholar]
  32. Perez-Perez G. I., Blaser M. J. Conservation and diversity of Campylobacter pyloridis major antigens. Infect Immun. 1987 May;55(5):1256–1263. doi: 10.1128/iai.55.5.1256-1263.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Skirrow M. B. Campylobacter enteritis: a "new" disease. Br Med J. 1977 Jul 2;2(6078):9–11. doi: 10.1136/bmj.2.6078.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Smoot D. T., Mobley H. L., Chippendale G. R., Lewison J. F., Resau J. H. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun. 1990 Jun;58(6):1992–1994. doi: 10.1128/iai.58.6.1992-1994.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES