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. 1995 Dec;177(24):7178–7185. doi: 10.1128/jb.177.24.7178-7185.1995

Purification of the copper response extracellular proteins secreted by the copper-resistant methanogen Methanobacterium bryantii BKYH and cloning, sequencing, and transcription of the gene encoding these proteins.

B K Kim 1, T D Pihl 1, J N Reeve 1, L Daniels 1
PMCID: PMC177598  PMID: 8522526

Abstract

When the copper-resistant methanogen Methanobacterium bryantii BKYH was exposed to 1 mM Cu(II), it secreted approximately fourfold increased levels of three proteins, copper response extracellular (CRX) proteins. The members of the CRX protein trio had apparent molecular masses of 40.8, 42.3, and 42.9 kDa and were purified together from the culture supernatant and separated from each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the three proteins were essentially identical, and antibodies raised against one of the trio reacted with all three proteins and with three other intracellular proteins with slightly higher molecular weights. The N-terminal amino acid sequence of one of these larger proteins was different from that of the secreted CRX proteins. The gene crx, which encodes the CRX proteins, was cloned and sequenced, and crx transcription was characterized. The crx sequence predicts that the encoded polypeptide is synthesized as a precursor with an N-terminal leader peptide, containing 28 amino acid residues, that is removed during the extracellular secretion of the CRX proteins. Transcription was initiated 274 bp upstream from the crx gene, producing an approximately 1.4-kb monocistronic transcript that was present in M. bryantii BKYH cells under all growth conditions but that increased approximately fourfold in vivo in response to Cu addition. The CRX proteins appear to be glycosylated, since they react with concanavalin A and neuraminidase, and to be the products of one gene that have different levels of posttranslational glycosylation. This is supported by very similar chromatographic and electrophoretic properties, identical N-terminal amino acid sequences, immunological cross-reactivities, and the detection of only one crx-related sequence by Southern blotting. Western blots (immunoblots) showed no evidence for CRX proteins in cell lysates of several other Methanobacterium strains.

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Selected References

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  1. Bender C. L., Cooksey D. A. Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance. J Bacteriol. 1986 Feb;165(2):534–541. doi: 10.1128/jb.165.2.534-541.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender C. L., Cooksey D. A. Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato. J Bacteriol. 1987 Feb;169(2):470–474. doi: 10.1128/jb.169.2.470-474.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender C. L., Malvick D. K., Conway K. E., George S., Pratt P. Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol. 1990 Jan;56(1):170–175. doi: 10.1128/aem.56.1.170-175.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brown N. L., Rouch D. A., Lee B. T. Copper resistance determinants in bacteria. Plasmid. 1992 Jan;27(1):41–51. doi: 10.1016/0147-619x(92)90005-u. [DOI] [PubMed] [Google Scholar]
  6. Bröckl G., Behr M., Fabry S., Hensel R., Kaudewitz H., Biendl E., König H. Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis. Eur J Biochem. 1991 Jul 1;199(1):147–152. doi: 10.1111/j.1432-1033.1991.tb16102.x. [DOI] [PubMed] [Google Scholar]
  7. Clarke S. E., Stuart J., Sanders-Loehr J. Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity. Appl Environ Microbiol. 1987 May;53(5):917–922. doi: 10.1128/aem.53.5.917-922.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooksey D. A., Azad H. R., Cha J. S., Lim C. K. Copper resistance gene homologs in pathogenic and saprophytic bacterial species from tomato. Appl Environ Microbiol. 1990 Feb;56(2):431–435. doi: 10.1128/aem.56.2.431-435.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooksey D. A. Characterization of a Copper Resistance Plasmid Conserved in Copper-Resistant Strains of Pseudomonas syringae pv. tomato. Appl Environ Microbiol. 1987 Feb;53(2):454–456. doi: 10.1128/aem.53.2.454-456.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cooksey D. A. Copper uptake and resistance in bacteria. Mol Microbiol. 1993 Jan;7(1):1–5. doi: 10.1111/j.1365-2958.1993.tb01091.x. [DOI] [PubMed] [Google Scholar]
  11. Davis G. K. High-level copper feeding of swine and poultry and the ecology. Fed Proc. 1974 May;33(5):1194–1196. [PubMed] [Google Scholar]
  12. Erardi F. X., Failla M. L., Falkinham J. O., 3rd Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum. Appl Environ Microbiol. 1987 Aug;53(8):1951–1954. doi: 10.1128/aem.53.8.1951-1954.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harwood-Sears V., Gordon A. S. Copper-induced production of copper-binding supernatant proteins by the marine bacterium Vibrio alginolyticus. Appl Environ Microbiol. 1990 May;56(5):1327–1332. doi: 10.1128/aem.56.5.1327-1332.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hennigan A. N., Reeve J. N. mRNAs in the methanogenic archaeon Methanococcus vannielii: numbers, half-lives and processing. Mol Microbiol. 1994 Feb;11(4):655–670. doi: 10.1111/j.1365-2958.1994.tb00344.x. [DOI] [PubMed] [Google Scholar]
  15. Kalmokoff M. L., Karnauchow T. M., Jarrell K. F. Conserved N-terminal sequences in the flagellins of archaebacteria. Biochem Biophys Res Commun. 1990 Feb 28;167(1):154–160. doi: 10.1016/0006-291x(90)91744-d. [DOI] [PubMed] [Google Scholar]
  16. Kim Byoung-Kwan, Daniels Lacy. Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria. Appl Environ Microbiol. 1991 Jun;57(6):1866–1869. doi: 10.1128/aem.57.6.1866-1869.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kiyohara T., Terao T., Shioiri-Nakano K., Osawa T. Purification and properties of a neuraminidase from Streptococcus K 6646. Arch Biochem Biophys. 1974 Oct;164(2):575–582. doi: 10.1016/0003-9861(74)90069-1. [DOI] [PubMed] [Google Scholar]
  18. Mittelman M. W., Geesey G. G. Copper-binding characteristics of exopolymers from a freshwater-sediment bacterium. Appl Environ Microbiol. 1985 Apr;49(4):846–851. doi: 10.1128/aem.49.4.846-851.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Montzka K. A., Steitz J. A. Additional low-abundance human small nuclear ribonucleoproteins: U11, U12, etc. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8885–8889. doi: 10.1073/pnas.85.23.8885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pihl T. D., Sharma S., Reeve J. N. Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H. J Bacteriol. 1994 Oct;176(20):6384–6391. doi: 10.1128/jb.176.20.6384-6391.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reeve J. N. Molecular biology of methanogens. Annu Rev Microbiol. 1992;46:165–191. doi: 10.1146/annurev.mi.46.100192.001121. [DOI] [PubMed] [Google Scholar]
  22. Silver S., Walderhaug M. Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria. Microbiol Rev. 1992 Mar;56(1):195–228. doi: 10.1128/mr.56.1.195-228.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tetaz T. J., Luke R. K. Plasmid-controlled resistance to copper in Escherichia coli. J Bacteriol. 1983 Jun;154(3):1263–1268. doi: 10.1128/jb.154.3.1263-1268.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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