Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Jul;173(14):4493–4502. doi: 10.1128/jb.173.14.4493-4502.1991

Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.

L Bissonnette 1, S Champetier 1, J P Buisson 1, P H Roy 1
PMCID: PMC208113  PMID: 1648560

Abstract

Integrons constitute a novel family of DNA elements which evolved by site-specific integration of discrete units between two conserved segments. On the In4 integron of Tn1696, a precisely inserted gene cassette of 1,549 bp conferring nonenzymatic chloramphenicol resistance (cmlA) is present between the streptomycin-spectinomycin resistance (aadA2) gene cassette and the 3'-conserved segment of the integron. In this study, we present the nucleotide sequence of the cmlA gene cassette of Tn1696, show its similarity to bacterial efflux systems and other transport proteins, and present evidence for alterations that its expression exerts on bacterial membranes. The cmlA gene cassette apparently carries its own promoter(s), a situation that has not heretofore been observed in the integrons of multiresistance plasmids and transposons of gram-negative bacteria. One or more of these promoters were shown to be functionally active in expressing a cat marker gene from promoter-probe vectors. The putative CmlA polypeptide appears to provoke a reduction of the content of the major porins OmpA and OmpC.

Full text

PDF
4497

Images in this article

Selected References

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

  1. Beaulieu D., Ouellette M., Bergeron M. G., Roy P. H. Characterization of a plasmid isolated from Branhamella catarrhalis and detection of plasmid sequences within the genome of a B. catarrhalis strain. Plasmid. 1988 Sep;20(2):158–162. doi: 10.1016/0147-619x(88)90020-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Burns J. L., Hedin L. A., Lien D. M. Chloramphenicol resistance in Pseudomonas cepacia because of decreased permeability. Antimicrob Agents Chemother. 1989 Feb;33(2):136–141. doi: 10.1128/aac.33.2.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burns J. L., Mendelman P. M., Levy J., Stull T. L., Smith A. L. A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae. Antimicrob Agents Chemother. 1985 Jan;27(1):46–54. doi: 10.1128/aac.27.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burns J. L., Rubens C. E., Mendelman P. M., Smith A. L. Cloning and expression in Escherichia coli of a gene encoding nonenzymatic chloramphenicol resistance from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1986 Mar;29(3):445–450. doi: 10.1128/aac.29.3.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S. P., McMurry L. M., Hooper D. C., Wolfson J. S., Levy S. B. Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrob Agents Chemother. 1989 Aug;33(8):1318–1325. doi: 10.1128/aac.33.8.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dick T., Matzura H. Chloramphenicol-induced translational activation of cat messenger RNA in vitro. J Mol Biol. 1990 Apr 20;212(4):661–668. doi: 10.1016/0022-2836(90)90228-E. [DOI] [PubMed] [Google Scholar]
  9. Dorman C. J., Foster T. J. Nonenzymatic chloramphenicol resistance determinants specified by plasmids R26 and R55-1 in Escherichia coli K-12 do not confer high-level resistance to fluorinated analogs. Antimicrob Agents Chemother. 1982 Nov;22(5):912–914. doi: 10.1128/aac.22.5.912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorman C. J., Foster T. J. Posttranscriptional regulation of the inducible nonenzymatic chloramphenicol resistance determinant of IncP plasmid R26. J Bacteriol. 1985 Jan;161(1):147–152. doi: 10.1128/jb.161.1.147-152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dorman C. J., Foster T. J., Shaw W. V. Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product. Gene. 1986;41(2-3):349–353. doi: 10.1016/0378-1119(86)90119-8. [DOI] [PubMed] [Google Scholar]
  12. Eckert B., Beck C. F. Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli. J Biol Chem. 1989 Jul 15;264(20):11663–11670. [PubMed] [Google Scholar]
  13. Fasman G. D., Gilbert W. A. The prediction of transmembrane protein sequences and their conformation: an evaluation. Trends Biochem Sci. 1990 Mar;15(3):89–92. doi: 10.1016/0968-0004(90)90187-g. [DOI] [PubMed] [Google Scholar]
  14. Forst S., Inouye M. Environmentally regulated gene expression for membrane proteins in Escherichia coli. Annu Rev Cell Biol. 1988;4:21–42. doi: 10.1146/annurev.cb.04.110188.000321. [DOI] [PubMed] [Google Scholar]
  15. Gaffney D. F., Cundliffe E., Foster T. J. Chloramphenicol resistance that does not involve chloramphenicol acetyltransferase encoded by plasmids from gram-negative bacteria. J Gen Microbiol. 1981 Jul;125(1):113–121. doi: 10.1099/00221287-125-1-113. [DOI] [PubMed] [Google Scholar]
  16. Godfrey A. J., Hatlelid L., Bryan L. E. Correlation between lipopolysaccharide structure and permeability resistance in beta-lactam-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1984 Aug;26(2):181–186. doi: 10.1128/aac.26.2.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  18. Guerineau F., Brooks L., Mullineaux P. Expression of the sulfonamide resistance gene from plasmid R46. Plasmid. 1990 Jan;23(1):35–41. doi: 10.1016/0147-619x(90)90042-b. [DOI] [PubMed] [Google Scholar]
  19. Hall R. M., Vockler C. The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. Nucleic Acids Res. 1987 Sep 25;15(18):7491–7501. doi: 10.1093/nar/15.18.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hirsch P. R., Wang C. L., Woodward M. J. Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033. Gene. 1986;48(2-3):203–209. doi: 10.1016/0378-1119(86)90078-8. [DOI] [PubMed] [Google Scholar]
  22. Iyobe S., Sagai H., Mitsuhashi S. Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa. J Bacteriol. 1981 Apr;146(1):141–148. doi: 10.1128/jb.146.1.141-148.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Komatsu T., Ohta M., Kido N., Arakawa Y., Ito H., Mizuno T., Kato N. Molecular characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of Escherichia coli outer membrane proteins. J Bacteriol. 1990 Jul;172(7):4082–4089. doi: 10.1128/jb.172.7.4082-4089.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kono M., O'Hara K. Mechanism of chloramphenicol-resistance mediated by kR102 factor in Pseudomonas aeruginosa. J Antibiot (Tokyo) 1976 Feb;29(2):176–180. doi: 10.7164/antibiotics.29.176. [DOI] [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. Lovett P. S. Translational attenuation as the regulator of inducible cat genes. J Bacteriol. 1990 Jan;172(1):1–6. doi: 10.1128/jb.172.1.1-6.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martinez E., de la Cruz F. Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J. 1990 Apr;9(4):1275–1281. doi: 10.1002/j.1460-2075.1990.tb08236.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martinez E., de la Cruz F. Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet. 1988 Feb;211(2):320–325. doi: 10.1007/BF00330610. [DOI] [PubMed] [Google Scholar]
  29. Meyer J. F., Nies B. A., Kratz J., Wiedemann B. Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161. J Gen Microbiol. 1985 May;131(5):1123–1130. doi: 10.1099/00221287-131-5-1123. [DOI] [PubMed] [Google Scholar]
  30. Meyer J. F., Nies B. A., Wiedemann B. Amikacin resistance mediated by multiresistance transposon Tn2424. J Bacteriol. 1983 Aug;155(2):755–760. doi: 10.1128/jb.155.2.755-760.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mosher R. H., Ranade N. P., Schrempf H., Vining L. C. Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae. J Gen Microbiol. 1990 Feb;136(2):293–301. doi: 10.1099/00221287-136-2-293. [DOI] [PubMed] [Google Scholar]
  32. Nagai Y., Mitsuhashi S. New type of R factors incapable of inactivating chloramphenicol. J Bacteriol. 1972 Jan;109(1):1–7. doi: 10.1128/jb.109.1.1-7.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neal R. J., Chater K. F. Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. Gene. 1987;58(2-3):229–241. doi: 10.1016/0378-1119(87)90378-7. [DOI] [PubMed] [Google Scholar]
  34. Nikaido H. Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob Agents Chemother. 1989 Nov;33(11):1831–1836. doi: 10.1128/aac.33.11.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ouellette M., Bissonnette L., Roy P. H. Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7378–7382. doi: 10.1073/pnas.84.21.7378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ouellette M., Roy P. H. Homology of ORFs from Tn2603 and from R46 to site-specific recombinases. Nucleic Acids Res. 1987 Dec 10;15(23):10055–10055. doi: 10.1093/nar/15.23.10055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Quigley N. B., Reeves P. R. Chloramphenicol resistance cloning vector based on pUC9. Plasmid. 1987 Jan;17(1):54–57. doi: 10.1016/0147-619x(87)90008-4. [DOI] [PubMed] [Google Scholar]
  38. Rouch D. A., Cram D. S., DiBerardino D., Littlejohn T. G., Skurray R. A. Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol. 1990 Dec;4(12):2051–2062. doi: 10.1111/j.1365-2958.1990.tb00565.x. [DOI] [PubMed] [Google Scholar]
  39. Rubens C. E., McNeill W. F., Farrar W. E., Jr Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents. J Bacteriol. 1979 Sep;139(3):877–882. doi: 10.1128/jb.139.3.877-882.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salyers A. A., Speer B. S., Shoemaker N. B. New perspectives in tetracycline resistance. Mol Microbiol. 1990 Jan;4(1):151–156. doi: 10.1111/j.1365-2958.1990.tb02025.x. [DOI] [PubMed] [Google Scholar]
  41. Sanders C. C., Sanders W. E., Jr, Goering R. V., Werner V. Selection of multiple antibiotic resistance by quinolones, beta-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrob Agents Chemother. 1984 Dec;26(6):797–801. doi: 10.1128/aac.26.6.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schmidt F. R., Nücken E. J., Henschke R. B. Nucleotide sequence analysis of 2''-aminoglycoside nucleotidyl-transferase ANT(2'') from Tn4000: its relationship with AAD(3'') and impact on Tn21 evolution. Mol Microbiol. 1988 Nov;2(6):709–717. doi: 10.1111/j.1365-2958.1988.tb00081.x. [DOI] [PubMed] [Google Scholar]
  44. Schmidt F. R., Nücken E. J., Henschke R. B. Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons. Mol Microbiol. 1989 Nov;3(11):1545–1555. doi: 10.1111/j.1365-2958.1989.tb00140.x. [DOI] [PubMed] [Google Scholar]
  45. Schmidt F., Klopfer-Kaul I. Evolutionary relationship between Tn21-like elements and pBP201, a plasmid from Klebsiella pneumoniae mediating resistance to gentamicin and eight other drugs. Mol Gen Genet. 1984;197(1):109–119. doi: 10.1007/BF00327930. [DOI] [PubMed] [Google Scholar]
  46. Shaw W. V. Bacterial resistance to chloramphenicol. Br Med Bull. 1984 Jan;40(1):36–41. doi: 10.1093/oxfordjournals.bmb.a071945. [DOI] [PubMed] [Google Scholar]
  47. Shaw W. V. Chloramphenicol acetyltransferase: enzymology and molecular biology. CRC Crit Rev Biochem. 1983;14(1):1–46. doi: 10.3109/10409238309102789. [DOI] [PubMed] [Google Scholar]
  48. Stokes H. W., Hall R. M. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol. 1989 Dec;3(12):1669–1683. doi: 10.1111/j.1365-2958.1989.tb00153.x. [DOI] [PubMed] [Google Scholar]
  49. Sundström L., Rådström P., Swedberg G., Sköld O. Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol Gen Genet. 1988 Aug;213(2-3):191–201. doi: 10.1007/BF00339581. [DOI] [PubMed] [Google Scholar]
  50. Tait R. C., Rempel H., Rodriguez R. L., Kado C. I. The aminoglycoside-resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene. Gene. 1985;36(1-2):97–104. doi: 10.1016/0378-1119(85)90073-3. [DOI] [PubMed] [Google Scholar]
  51. Tenover F. C., Filpula D., Phillips K. L., Plorde J. J. Cloning and sequencing of a gene encoding an aminoglycoside 6'-N-acetyltransferase from an R factor of Citrobacter diversus. J Bacteriol. 1988 Jan;170(1):471–473. doi: 10.1128/jb.170.1.471-473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tenover F. C., Phillips K. L., Gilbert T., Lockhart P., O'Hara P. J., Plorde J. J. Development of a DNA probe from the deoxyribonucleotide sequence of a 3-N-aminoglycoside acetyltransferase [AAC(3)-I] resistance gene. Antimicrob Agents Chemother. 1989 Apr;33(4):551–559. doi: 10.1128/aac.33.4.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Toro C. S., Lobos S. R., Calderón I., Rodríguez M., Mora G. C. Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol. Antimicrob Agents Chemother. 1990 Sep;34(9):1715–1719. doi: 10.1128/aac.34.9.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Villarroel R., Hedges R. W., Maenhaut R., Leemans J., Engler G., Van Montagu M., Schell J. Heteroduplex analysis of P-plasmid evolution: the role of insertion and deletion of transposable elements. Mol Gen Genet. 1983;189(3):390–399. doi: 10.1007/BF00325900. [DOI] [PubMed] [Google Scholar]
  55. Wiedemann B., Meyer J. F., Zühlsdorf M. T. Insertions of resistance genes into Tn21-like transposons. J Antimicrob Chemother. 1986 Oct;18 (Suppl 100):85–92. doi: 10.1093/jac/18.supplement_c.85. [DOI] [PubMed] [Google Scholar]
  56. Wohlleben W., Arnold W., Bissonnette L., Pelletier A., Tanguay A., Roy P. H., Gamboa G. C., Barry G. F., Aubert E., Davies J. On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette. Mol Gen Genet. 1989 Jun;217(2-3):202–208. doi: 10.1007/BF02464882. [DOI] [PubMed] [Google Scholar]
  57. Yoshida H., Bogaki M., Nakamura S., Ubukata K., Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990 Dec;172(12):6942–6949. doi: 10.1128/jb.172.12.6942-6949.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES