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, DH5α, 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.
Chang, A. C. & Cohen, S. N.(1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from P15A cryptic miniplasmid. J Bacteriol134, 1141–1156.
[Google Scholar]
Chen, Y. Y., Weaver, C. A. & Burne, R. A.(2000). Dual functions of Streptococcus salivarius urease. J. Bacteriol182, 4667–4669.[CrossRef][Google Scholar]
Griffin, P. M. & Tauxe, R. V.(1991). The epidemiology of infections caused by Escherichia coli O157 : H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev13, 60–98.
[Google Scholar]
Gyles, C., Johnson, R., Gao, A., Ziebell, K., Pierard, D., Aleksic, S. & Boerlin, P.(1998). Association of enterohemorrhagic Escherichia coli of human and bovine origins. Appl Environ Microbiol64, 4134–4141.
[Google Scholar]
Hansen, L. M. & Solnick, J. V.(2001). Selection for urease activity during Helicobacter pylori infection of rhesus macaques (Macaca mulatta). Infect Immun69, 3519–3522.[CrossRef][Google Scholar]
Hayashi, T., Makino, K., Ohnishi, M. & 19 other authors(2001). Complete genome sequence of enterohemorrhagic Escherichia coli O157 : H7 and genomic comparison with a laboratory strain K-12. DNA Res8, 11–22.[CrossRef][Google Scholar]
Heimer, S. R., Welch, R. A., Perna, N. T., Posfai, G., Evans, P. S., Kaper, J. B., Blattner, F. R. & Mobley, H. L. T.(2002). Urease of enterohemorrhagic Escherichia coli: evidence for regulation by Fur and a trans-acting factor. Infect Immun70, 1027–1031.[CrossRef][Google Scholar]
Lee, M. H., Mulrooney, S. B., Renner, M. J., Markowicz, Y. & Hausinger, R. P.(1992).Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J Bacteriol174, 4324–4330.
[Google Scholar]
Makino, K., Ishii, K., Yasunaga, T. & 14 other authors(1998). Complete nucleotide sequences of 93-kb and 3·3-kb plasmids of an enterohemorrhagic Escherichia coli O157 : H7 derived from Sakai outbreak. DNA Res5, 1–9.[CrossRef][Google Scholar]
Makino, S. T., Tobe, H., Asakura, M., Watarai, T., Ikeda, K., Takeshi, & Sasakawa, C.(2003). Distribution of the secondary type III secretion system locus found in enterohemorrhagic Escherichia coli O157 : H7 isolated among Shiga toxin-producing E. coli strains. J Clin Microbiol41, 2341–2347.[CrossRef][Google Scholar]
McDaniel, T. K. & Kaper, J. B.(1997). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol23, 399–407.[CrossRef][Google Scholar]
Miller, J. H.(1972).Experiments in Molecular Genetics, pp. 173–179. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mobley, H. L. T., Island, M. D. & Hausinger, R. P.(1995). Molecular biology of microbial ureases. Microbiol Rev59, 451–480.
[Google Scholar]
Mulrooney, S. B. & Hausinger, R. P.(1990). Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J Bacteriol172, 5837–5843.
[Google Scholar]
Nakano, M., Iida, T., Ohnishi, M., Kurokawa, K., Takahashi, A., Tsukamoto, T., Yasunaga, T., Hayashi, T. & Honda, T.(2001). Association of the urease gene with enterohemorrhagic Escherichia coli strains irrespective of their serogroups. J Clin Microbiol39, 4541–4543.[CrossRef][Google Scholar]
Park, I.-S. & Hausinger, R. P.(1995). Requirement of CO2 for in vitro assembly of the urease nickel metallocenter. Science267, 1156–1158.[CrossRef][Google Scholar]
Park, I.-S., Carr, M. B. & Hausinger, R. P.(1994).In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. Proc Natl Acad Sci U S A91, 3233–3237.[CrossRef][Google Scholar]
Park, K. S., Iida, T., Yamaichi, Y., Ohyagi, T., Yamamoto, K. & Honda, T.(2000). Genetic characterization of DNA region containing the trh and ure genes of Vibrio parahaemolyticus. Infect Immun68, 5742–5748.[CrossRef][Google Scholar]
Perna, N. T., Mayhew, G. F., Posfai, G., Elliott, S., Donnenberg, M. S., Kaper, J. B. & Blattner, F. R.(1998). Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157 : H7. Infect Immun66, 3810–3817.
[Google Scholar]
Perna, N. T., Plunkett, G., 3rd, Bruland, V. & 24 other authors(2001). Genome sequence of enterohemorrhagic Escherichia coli O157 : H7. Nature409, 529–533.[CrossRef][Google Scholar]
Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989). Enzymes used in molecular cloning. In Molecular Cloning: a Laboratory Manual, 2nd edn, pp. 5.3–5.86. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sebbane, F., Devalckenere, A., Foulon, J., Carniel, E. & Simonet, M.(2001). Silencing and reactivity of urease in Yersinia pestis is determined by one G residue at a specific position in the ureD gene. Infect Immun69, 170–176.[CrossRef][Google Scholar]
Tutenel, A. V., Pierard, D., Van Hoof, J., Cornelis, M. & De Zutter, L.(2003). Isolation and molecular characterization of Escherichia coli O157 isolated from cattle, pigs, and chickens at slaughter. Int J Food Microbiol84, 63–69.[CrossRef][Google Scholar]
Verweyen, H. M., Harch, H., Brandis, M. & Zimmerhackl, L. B.(2000). Enterohemorrhagic Escherichia coli infections: following transmission routes. Pediatr Nephrol14, 73–87.[CrossRef][Google Scholar]
Watanabe, H., Wada, A., Inagaki, Y., Itoh, K. & Tamura, K.(1996). Outbreaks of enterohaemorrhagic Escherichia coli O157 : H7 infections by two different genotype strains in Japan. Lancet348, 831–832.[CrossRef][Google Scholar]
Wilson, K.(1994). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, vol. 1, 8th edn, pp. 2.4.1–2.4.5. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York, NY: Current Protocols.
Yanisch-Perron, C., Vieira, J. & Messing, J.(1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33, 103–119.[CrossRef][Google Scholar]