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. 1984 Nov;160(2):556–563. doi: 10.1128/jb.160.2.556-563.1984

Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans.

J A Tobian, M L Cline, F L Macrina
PMCID: PMC214770  PMID: 6094475

Abstract

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.

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Selected References

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  1. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  2. Burdett V. Identification of tetracycline-resistant R-plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother. 1980 Nov;18(5):753–760. doi: 10.1128/aac.18.5.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burdett V., Inamine J., Rajagopalan S. Heterogeneity of tetracycline resistance determinants in Streptococcus. J Bacteriol. 1982 Mar;149(3):995–1004. doi: 10.1128/jb.149.3.995-1004.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chopra I., Howe T. G. Bacterial resistance to the tetracyclines. Microbiol Rev. 1978 Dec;42(4):707–724. doi: 10.1128/mr.42.4.707-724.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  6. Clewell D. B. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol Rev. 1981 Sep;45(3):409–436. doi: 10.1128/mr.45.3.409-436.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crooks J. H., Ullman M., Zoller M., Levy S. B. Transcription of plasmid DNA in Escherichia coli minicells. Plasmid. 1983 Jul;10(1):66–72. doi: 10.1016/0147-619x(83)90058-6. [DOI] [PubMed] [Google Scholar]
  8. Eccles S., Docherty A., Chopra I., Shales S., Ball P. Tetracycline resistance genes from Bacillus plasmid pAB124 confer decreased accumulation of the antibiotic in Bacillus subtilis but not in Escherichia coli. J Bacteriol. 1981 Mar;145(3):1417–1420. doi: 10.1128/jb.145.3.1417-1420.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Franke A. E., Clewell D. B. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J Bacteriol. 1981 Jan;145(1):494–502. doi: 10.1128/jb.145.1.494-502.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gayda R. C., Tanabe J. H., Knigge K. M., Markovitz A. Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance. Plasmid. 1979 Jul;2(3):417–425. doi: 10.1016/0147-619x(79)90025-8. [DOI] [PubMed] [Google Scholar]
  11. Hansen J. B., Olsen R. H. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J Bacteriol. 1978 Jul;135(1):227–238. doi: 10.1128/jb.135.1.227-238.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hawley R. J., Lee L. N., LeBlanc D. J. Effects of tetracycline on the streptococcal flora of periodontal pockets. Antimicrob Agents Chemother. 1980 Mar;17(3):372–378. doi: 10.1128/aac.17.3.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kennedy N., Beutin L., Achtman M., Skurray R., Rahmsdorf U., Herrlich P. Conjugation proteins encoded by the F sex factor. Nature. 1977 Dec 15;270(5638):580–585. doi: 10.1038/270580a0. [DOI] [PubMed] [Google Scholar]
  14. Lawson J. W., Gooder H. Growth and development of competence in the group H streptococci. J Bacteriol. 1970 Jun;102(3):820–825. doi: 10.1128/jb.102.3.820-825.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levy S. B., McMurry L. Detection of an inducible membrane protein associated with R-factor-mediated tetracycline resistance. Biochem Biophys Res Commun. 1974 Feb 27;56(4):1060–1068. doi: 10.1016/s0006-291x(74)80296-2. [DOI] [PubMed] [Google Scholar]
  16. Macrina F. L., Jones K. R., Wood P. H. Chimeric streptococcal plasmids and their use as molecular cloning vehicles in Streptococcus sanguis (Challis). J Bacteriol. 1980 Sep;143(3):1425–1435. doi: 10.1128/jb.143.3.1425-1435.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Macrina F. L., Kopecko D. J., Jones K. R., Ayers D. J., McCowen S. M. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid. 1978 Jun;1(3):417–420. doi: 10.1016/0147-619x(78)90056-2. [DOI] [PubMed] [Google Scholar]
  18. Macrina F. L., Tobian J. A., Jones K. R., Evans R. P., Clewell D. B. A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene. 1982 Oct;19(3):345–353. doi: 10.1016/0378-1119(82)90025-7. [DOI] [PubMed] [Google Scholar]
  19. Macrina F. L., Weatherly G. G., Curtiss R., 3rd R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12. J Bacteriol. 1974 Dec;120(3):1387–1400. doi: 10.1128/jb.120.3.1387-1400.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McMurry L., Petrucci R. E., Jr, Levy S. B. Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3974–3977. doi: 10.1073/pnas.77.7.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mendez B., Tachibana C., Levy S. B. Heterogeneity of tetracycline resistance determinants. Plasmid. 1980 Mar;3(2):99–108. doi: 10.1016/0147-619x(80)90101-8. [DOI] [PubMed] [Google Scholar]
  22. Meyers J. A., Sanchez D., Elwell L. P., Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol. 1976 Sep;127(3):1529–1537. doi: 10.1128/jb.127.3.1529-1537.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mooi F. R., Harms N., Bakker D., de Graaf F. K. Organization and expression of genes involved in the production of the K88ab antigen. Infect Immun. 1981 Jun;32(3):1155–1163. doi: 10.1128/iai.32.3.1155-1163.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  25. Robeson J. P., Barletta R. G., Curtiss R., 3rd Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli. J Bacteriol. 1983 Jan;153(1):211–221. doi: 10.1128/jb.153.1.211-221.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shales S. W., Chopra I., Ball P. R. Evidence for more than one mechanism of plasmid-determined tetracycline resistance in Escherichia coli. J Gen Microbiol. 1980 Nov;121(1):221–229. doi: 10.1099/00221287-121-1-221. [DOI] [PubMed] [Google Scholar]
  27. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  28. Tobian J. A., Macrina F. L. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome. J Bacteriol. 1982 Oct;152(1):215–222. doi: 10.1128/jb.152.1.215-222.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Winter R. B., Gold L. Overproduction of bacteriophage Q beta maturation (A2) protein leads to cell lysis. Cell. 1983 Jul;33(3):877–885. doi: 10.1016/0092-8674(83)90030-2. [DOI] [PubMed] [Google Scholar]
  30. Wojdani A., Avtalion R. R., Sompolinsky D. Isolation and characterization of tetracycline resistance proteins from Staphylococcus aureus and Escherichia coli. Antimicrob Agents Chemother. 1976 Mar;9(3):526–534. doi: 10.1128/aac.9.3.526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yagi Y., Clewell D. B. Amplification of the tetracycline resistance determinant of plasmid pAM alpha 1 in Streptococcus faecalis: dependence on host recombination machinery. J Bacteriol. 1980 Aug;143(2):1070–1072. doi: 10.1128/jb.143.2.1070-1072.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

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