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. 1993 Oct;175(20):6492–6498. doi: 10.1128/jb.175.20.6492-6498.1993

Characterization and transcriptional regulation of the 2'-N-acetyltransferase gene from Providencia stuartii.

P N Rather 1, E Orosz 1, K J Shaw 1, R Hare 1, G Miller 1
PMCID: PMC206758  PMID: 8407825

Abstract

We have cloned the chromosomally encoded 2'-N-acetyltransferase gene [aac(2')-Ia] from Providencia stuartii. DNA sequence analysis of the cloned insert identified a single open reading frame, which is capable of encoding a protein with a predicted molecular mass of 20,073 Da. The deduced AAC(2')-Ia protein showed no significant homology to other proteins, including all of the AAC(3) and AAC(6') proteins. Primer extension analysis was used to identify the aac(2')-Ia promoter, which contained an unusual sequence (CTTTTT) at the -35 region. Expression of the aac(2')-Ia gene occurs at low levels in wild-type P. stuartii strains; therefore, they are aminoglycoside susceptible. We have isolated mutants with high-level AAC(2')-Ia expression at a frequency of 4.8 x 10(-6). Detailed analysis of one mutant demonstrated a 12.2-fold increase in the accumulation of aac(2')-Ia mRNA. In addition, the levels of beta-galactosidase expression from a plasmid-encoded aac(2')-lacZ transcriptional fusion were increased 11.5-fold in this mutant relative to those in an isogenic wild-type strain. These results suggested that a trans-acting factor, designated aar (for aminoglycoside acetyltransferase regulator), controls AAC(2')-Ia expression in P. stuartii.

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Selected References

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  1. 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]
  2. Champion H. M., Bennett P. M., Lewis D. A., Reeves D. S. Cloning and characterization of an AAC(6') gene from Serratia marcescens. J Antimicrob Chemother. 1988 Nov;22(5):587–596. doi: 10.1093/jac/22.5.587. [DOI] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chevereau M., Daniels P. J., Davies J., LeGoffic F. Aminoglycoside resistance in bacteria medicated by gentamicin acetyltransferase II, an enzyme modifying the 2'-amino group of aminoglycoside antibiotics. Biochemistry. 1974 Jan 29;13(3):598–603. doi: 10.1021/bi00700a030. [DOI] [PubMed] [Google Scholar]
  5. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davies J., Smith D. I. Plasmid-determined resistance to antimicrobial agents. Annu Rev Microbiol. 1978;32:469–518. doi: 10.1146/annurev.mi.32.100178.002345. [DOI] [PubMed] [Google Scholar]
  8. Dupont C., Clarke A. J. Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis. J Bacteriol. 1991 Jul;173(14):4318–4324. doi: 10.1128/jb.173.14.4318-4324.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helmann J. D., Chamberlin M. J. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6422–6424. doi: 10.1073/pnas.84.18.6422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  14. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  15. John J. F., Jr, McNeill W. F., Price K. E., Kresel P. A. Evidence for a chromosomal site specifying amikacin resistance in multiresistant Serratia marcescens. Antimicrob Agents Chemother. 1982 Apr;21(4):587–591. doi: 10.1128/aac.21.4.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lange R., Hengge-Aronis R. Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S. J Bacteriol. 1991 Jul;173(14):4474–4481. doi: 10.1128/jb.173.14.4474-4481.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McHale P. J., Keane C. T., Dougan G. Antibiotic resistance in Providencia stuartii isolated in hospitals. J Clin Microbiol. 1981 Jun;13(6):1099–1104. doi: 10.1128/jcm.13.6.1099-1104.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rather P. N., Coppolecchia R., DeGrazia H., Moran C. P., Jr Negative regulator of sigma G-controlled gene expression in stationary-phase Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):709–715. doi: 10.1128/jb.172.2.709-715.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaw K. J., Rather P. N., Hare R. S., Miller G. H. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev. 1993 Mar;57(1):138–163. doi: 10.1128/mr.57.1.138-163.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shaw K. J., Rather P. N., Sabatelli F. J., Mann P., Munayyer H., Mierzwa R., Petrikkos G. L., Hare R. S., Miller G. H., Bennett P. Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens. Antimicrob Agents Chemother. 1992 Jul;36(7):1447–1455. doi: 10.1128/aac.36.7.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Swiatlo E., Kocka F. E. Inducible expression of an aminoglycoside-acetylating enzyme in Providencia stuartii. J Antimicrob Chemother. 1987 Jan;19(1):27–30. doi: 10.1093/jac/19.1.27. [DOI] [PubMed] [Google Scholar]
  22. Tanaka S., Matsushita Y., Yoshikawa A., Isono K. Cloning and molecular characterization of the gene rimL which encodes an enzyme acetylating ribosomal protein L12 of Escherichia coli K12. Mol Gen Genet. 1989 Jun;217(2-3):289–293. doi: 10.1007/BF02464895. [DOI] [PubMed] [Google Scholar]
  23. White-Ziegler C. A., Low D. A. Thermoregulation of the pap operon: evidence for the involvement of RimJ, the N-terminal acetylase of ribosomal protein S5. J Bacteriol. 1992 Nov;174(21):7003–7012. doi: 10.1128/jb.174.21.7003-7012.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yamaguchi M., Mitsuhashi S., Kobayashi F., Zenda H. A 2'-N-acetylating enzyme of aminoglycosides. J Antibiot (Tokyo) 1974 Jul;27(7):507–515. doi: 10.7164/antibiotics.27.507. [DOI] [PubMed] [Google Scholar]
  25. Yoshikawa A., Isono S., Sheback A., Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12. Mol Gen Genet. 1987 Oct;209(3):481–488. doi: 10.1007/BF00331153. [DOI] [PubMed] [Google Scholar]
  26. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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