1887

Abstract

A copper-transport () operon was cloned from the oral bacterium JH1005. DNA sequencing showed that the operon contained three genes (, and ), which were flanked by a single promoter and a factor-independent terminator. encoded a small protein of 147 aa with a heavy-metal-binding motif (CXCXCXC) at the C-terminus. CopY shared extensive homology with other bacterial negative transcriptional regulators. encoded a 742 aa protein that shared extensive homology with P-type ATPases. encoded a 67 aa protein that also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Northern blotting showed that a 32 kb transcript was produced by Cu-induced cells, suggesting that the genes were synthesized as a polycistronic message. The transcriptional start site of the operon was mapped and shown to lie within the inverted repeats of the promoter–operator region. wild-type cells were resistant to 800 μM Cu, whereas cells of a knock-out mutant were killed by 200 μM Cu. Complementation of the knock-out mutant with the operon restored Cu resistance to wild-type level. The wild-type and the mutant did not show any differences in susceptibility to other heavy metals, suggesting that the operon was specific for copper. By using a chloramphenicol acetyltransferase reporter gene fusion, the operon was shown to be negatively regulated by CopY and could be derepressed by Cu.

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2001-03-01
2021-07-30
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References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. E. 1990 Current Protocols in Molecular Biology. New York: Greene Publishing Associates/Wiley Interscience;
    [Google Scholar]
  2. Axelsen K. B., Palmgren M. G. 1998; Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46:84–101 [CrossRef]
    [Google Scholar]
  3. Bayle D., Wangler S., Weitzenegger T., Steinhilber W., Volz J., Przybylski M., Schafer K. P., Sachs G., Melchers K. 1998; Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis . J Bacteriol 180:317–329
    [Google Scholar]
  4. Cervantes C., Gutierrez-Corona F. 1994; Copper resistance mechanisms in bacteria and fungi. FEMS Microbiol Rev 14:121–137
    [Google Scholar]
  5. Cobine P., Wickramasinghe W. A., Harrison M. D., Weber T., Solioz M., Dameron C. T. 1999; The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor. FEBS Lett 445:27–30 [CrossRef]
    [Google Scholar]
  6. Dunny G. M., Lee L. N., LeBlanc D. J. 1991; Improved electroporation and cloning vector system for gram-positive bacteria . Appl Environ Microbiol 57:1194–1201
    [Google Scholar]
  7. Endo G., Silver S. 1995; CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258. J Bacteriol 177:4437–4441
    [Google Scholar]
  8. Ge Z., Hiratsuka K., Taylor D. E. 1995; Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport. Mol Microbiol 15:97–106 [CrossRef]
    [Google Scholar]
  9. Hiramatsu K., Asada K., Suzuki E., Okonogi K., Yokota T. 1992; Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus . FEBS Lett 298:13–136
    [Google Scholar]
  10. Homonylo-McGavin M. K., Lee S. F. 1996; Role of the C terminus in antigen P1 surface localization in Streptococcus mutans and two related cocci. J Bacteriol 178:801–807
    [Google Scholar]
  11. Hudson M. C., Stewart G. C. 1986; Differential utilization of Staphylococcus aureus promoter sequences by Escherichia coli and Bacillus subtilis . Gene 48:93–100 [CrossRef]
    [Google Scholar]
  12. Jenkinson H. F., Baker R. A., Tannock G. W. 1996; A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J Bacteriol 178:68–77
    [Google Scholar]
  13. Kroczek R. A., Siebert E. 1990; Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal Biochem 184:90–95 [CrossRef]
    [Google Scholar]
  14. Lamond A. I., Travers A. A. 1983; Requirement for an upstream element for optimal transcription of a bacterial tRNA gene. Nature 305:248–250 [CrossRef]
    [Google Scholar]
  15. Lee S. F., Progulske-Fox A., Bleiweis A. S. 1988; Molecular cloning and expression of a Streptococcus mutans major surface protein antigen,P1 (I/II), in Escherichia coli . Infect Immun 56:2114–2119
    [Google Scholar]
  16. Loesche W. J. 1986; Role of Streptococcus mutans in human dental decay. Microbiol Rev 50:353–380
    [Google Scholar]
  17. Lunsford R. D. 1995; Recovery of RNA from oral streptococci. Biotechniques 18:412–414
    [Google Scholar]
  18. Morrier J. J., Suchett-Kaye G., Nguyen D., Rocca J. P., Blanc-Benon J., Barsotti O. 1998; Antimicrobial activity of amalgams, alloys and their elements and phases. Dent Mater 14:150–157 [CrossRef]
    [Google Scholar]
  19. Nucifora G., Chu L., Misra T. K., Silver S. 1989; Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase. Proc Natl Acad Sci USA 86:3544–3548 [CrossRef]
    [Google Scholar]
  20. Odermatt A., Solioz M. 1995; Two trans-acting metalloregulatory proteins controlling expression of the copper-ATPases of Enterococcus hirae . J Biol Chem 270:4349–4354 [CrossRef]
    [Google Scholar]
  21. Odermatt A., Suter H., Krapf R., Solioz M. 1993; Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae . J Biol Chem 268:12775–12779
    [Google Scholar]
  22. Payne A. S., Gitlin J. D. 1998; Functional expression of the menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases. J Biol Chem 273:3765–3770 [CrossRef]
    [Google Scholar]
  23. Petrukhin K., Lutsenko S., Chernov I., Ross B. M., Kaplan J. H., Gilliam T. C. 1994; Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions . Hum Mol Genet 3:1647–1656 [CrossRef]
    [Google Scholar]
  24. Rensing C., Ghosh M., Rosen B. P. 1999; Families of soft-metal-ion-transporting ATPases. J Bacteriol 181:5891–5897
    [Google Scholar]
  25. Rensing C., Fan B., Sharma R., Mitra B., Rosen B. P. 2000; CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci USA 97:652–656 [CrossRef]
    [Google Scholar]
  26. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Shaw J. H., Clewell D. B. 1985; Complete nucleotide sequence of macrolide–lincosamide–streptogramin B-resistance transposon Tn 917 in Streptococcus faecalis. J Bacteriol 164:782–796
    [Google Scholar]
  28. Silver S., Phung L. T. 1996; Bacterial heavy metal resistance: new surprises . Annu. Rev Microbiol 50:753–789 [CrossRef]
    [Google Scholar]
  29. Smith M. C., Murray B. E. 1992; Sequence analysis of the β-lactamase repressor from Staphylococcus aureus and hybridization studies with two β-lactamase-producing isolates of Enterococcus faecalis . Antimicrob Agents Chemother 36:2265–2269 [CrossRef]
    [Google Scholar]
  30. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517 [CrossRef]
    [Google Scholar]
  31. Strausak D., Solioz M. 1997; CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases. J Biol Chem 272:8932–8936 [CrossRef]
    [Google Scholar]
  32. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [CrossRef]
    [Google Scholar]
  33. Tobian J. A., Cline M. L., Macrina F. L. 1984; Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans. J Bacteriol 160:556–563
    [Google Scholar]
  34. Vats N., Lee S. F. 2000; Active detachment of Streptococcus mutans cells adhered to epon-hydroxylapatite surfaces coated with salivary proteins in vitro. Arch Oral Biol 45:305–314 [CrossRef]
    [Google Scholar]
  35. Wataha J. C., Lockwood P. E. 1998; Release of elements from dental casting alloys into cell-culture medium over 10 months. Dent Mater 14:158–163 [CrossRef]
    [Google Scholar]
  36. Wittman V., Lin H. C., Wong H. C. 1993; Functional domains of the penicillinase repressor of Bacillus licheniformis. J Bacteriol 175:7383–7390
    [Google Scholar]
  37. Wunderli-Ye H., Solioz M. 1999a; Copper homeostasis in Enterococcus hirae. Adv Epx Med Biol. 448255–264
  38. Wunderli-Ye H., Solioz M. 1999b; Effects of promoter mutations on the in vivo regulation of the cop operon of Enterococcus hirae by copper(I) & copper(II ). Biochem Biophys Res Commun. 259443–449 [CrossRef]
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