Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1991 May;35(5):892–897. doi: 10.1128/aac.35.5.892

Nucleotide sequence of the aacC3 gene, a gentamicin resistance determinant encoding aminoglycoside-(3)-N-acetyltransferase III expressed in Pseudomonas aeruginosa but not in Escherichia coli.

J S Vliegenthart 1, P A Ketelaar-van Gaalen 1, J A van de Klundert 1
PMCID: PMC245125  PMID: 1649572

Abstract

A chromosomal gentamicin resistance determinant from Pseudomonas aeruginosa was cloned on a 2.4-kb fragment in the broad-host-range vector pLAFR3. Substrate profiles for eight aminoglycosides at three concentrations showed that resistance was due to aminoglycoside-(3)-N-acetyltransferase III. This enzyme was produced in Pseudomonas strains but not in an Escherichia coli strain bearing the aacC3 gene. Nucleotide sequencing revealed two contiguous open reading frames (ORFs) preceded by a potential promoter and a ribosome-binding site. ORF-1 was 642 bp in length and encoded a protein of unknown function with a molecular mass of 23.9 kDa. ORF-2 was 813 bp in length and encoded a protein of 29.6 kDa. From deletion mutagenesis, in vitro transcription-translation data, and protein analysis of bacterial lysates, it was inferred that this 29.6-kDa protein represents the aminoglycoside-(3)-N-acetyltransferase III enzyme. A polymerase chain reaction with two specific intragenic 20-mer primers was developed to detect the aacC3 gene. A BstEII restriction site in the amplified DNA region was used to demonstrate the specificity of the reaction. Tests of 23 reference strains, which produced 12 different aminoglycoside-modifying enzymes, confirmed the specificities of the primers. The gene proved to be absent from a collection of 50 gentamicin-resistant P. aeruginosa strains selected at random in The Netherlands.

Full text

PDF
892

Images in this article

896Image
on p.896
896Image
on p.896

Selected References

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

  1. Allmansberger R., Bräu B., Piepersberg W. Genes for gentamicin-(3)-N-acetyl-transferases III and IV. II. Nucleotide sequences of three AAC(3)-III genes and evolutionary aspects. Mol Gen Genet. 1985;198(3):514–520. doi: 10.1007/BF00332949. [DOI] [PubMed] [Google Scholar]
  2. Bagdasarian M., Lurz R., Rückert B., Franklin F. C., Bagdasarian M. M., Frey J., Timmis K. N. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene. 1981 Dec;16(1-3):237–247. doi: 10.1016/0378-1119(81)90080-9. [DOI] [PubMed] [Google Scholar]
  3. Barg N. L. Construction of a probe for the aminoglycoside 3-V-acetyltransferase gene and detection of the gene among endemic clinical isolates. Antimicrob Agents Chemother. 1988 Dec;32(12):1834–1838. doi: 10.1128/aac.32.12.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biddlecome S., Haas M., Davies J., Miller G. H., Rane D. F., Daniels P. J. Enzymatic modification of aminoglycoside antibiotics: a new 3-N-acetylating enzyme from a Pseudomonas aeruginosa isolate. Antimicrob Agents Chemother. 1976 Jun;9(6):951–955. doi: 10.1128/aac.9.6.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bongaerts G. P., Vliegenthart J. S. Effect of aminoglycoside concentration on reaction rates of aminoglycoside-modifying enzymes. Antimicrob Agents Chemother. 1988 May;32(5):740–746. doi: 10.1128/aac.32.5.740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haas M. J., Dowding J. E. Aminoglycoside-modifying enzymes. Methods Enzymol. 1975;43:611–628. doi: 10.1016/0076-6879(75)43124-x. [DOI] [PubMed] [Google Scholar]
  9. Kettner M., Navarová J., Langsádl L. Aminoglycoside resistance patterns in clinical isolates of Enterobacteriaceae from Czechoslovakia. J Antimicrob Chemother. 1987 Sep;20(3):383–387. doi: 10.1093/jac/20.3.383. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. López-Cabrera M., Pérez-González J. A., Heinzel P., Piepersberg W., Jiménez A. Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene from Streptomyces rimosus forma paromomycinus. J Bacteriol. 1989 Jan;171(1):321–328. doi: 10.1128/jb.171.1.321-328.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maes P. Evaluation of the resistance mechanisms of gentamicin-resistant Gram-negative bacilli and their susceptibility to tobramycin, netilmicin and amikacin. J Antimicrob Chemother. 1985 Mar;15(3):283–289. doi: 10.1093/jac/15.3.283. [DOI] [PubMed] [Google Scholar]
  13. Miller J. H., Ganem D., Lu P., Schmitz A. Genetic studies of the lac repressor. I. Correlation of mutational sites with specific amino acid residues: construction of a colinear gene-protein map. J Mol Biol. 1977 Jan 15;109(2):275–298. doi: 10.1016/s0022-2836(77)80034-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Vliegenthart J. S., Ketelaar-van Gaalen P. A., van de Klundert J. A. Identification of three genes coding for aminoglycoside-modifying enzymes by means of the polymerase chain reaction. J Antimicrob Chemother. 1990 May;25(5):759–765. doi: 10.1093/jac/25.5.759. [DOI] [PubMed] [Google Scholar]
  18. Vliegenthart J. S., Ketelaar-van Gaalen P. A., van de Klundert J. A. Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae. Antimicrob Agents Chemother. 1989 Aug;33(8):1153–1159. doi: 10.1128/aac.33.8.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES