JAC Advance Access originally published online on November 20, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):39-45; doi:10.1093/jac/dkm440
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Original research |
Transcriptional analysis of and resistance level conferred by the aminoglycoside acetyltransferase gene aac(2')-Id from Mycobacterium smegmatis
1 Departamento de Microbiología, Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de Zaragoza, C/Domingo Miral s/n, 50009 Zaragoza, Spain 2 CIBER Enfermedades Respiratorias, Spain 3 Departamento de Medicina Preventiva, Salud Pública y Microbiología, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029 Madrid, Spain
Received 9 July 2007; returned 5 September 2007; revised 8 October 2007; accepted 18 October 2007
* Corresponding author. Tel: +34-976-762420; Fax: +34-976-762604; E-mail: ainsa{at}unizar.es
Objectives: To analyse the correlation between the expression levels of the aac(2')-Id gene from Mycobacterium smegmatis mc2155 and the resistance levels to aminoglycosides conferred by the encoded aminoglycoside 2'-N-acetyltransferase [AAC(2')-Id].
Methods: Expression levels were studied using a transductional fusion with the lacZ gene. The promoter region was characterized by primer extension analysis and ribonuclease protection assay. The aac(2')-Id gene was placed under the control of different mycobacterial promoters; deletions of the promoter region were done. Each of the plasmids was introduced in M. smegmatis mc2155 and the MICs were determined by resazurin assay.
Results: The aac(2')-Id gene is transcribed from two promoters: P1 (weaker) and P2 (stronger) located 38 and 1 nt upstream of the start codon, respectively. P2 promoter (producing a leaderless mRNA) was confirmed by producing deletions in the aac(2')-Id promoter and analysing the ability of the re-constructed genes to confer resistance to aminoglycosides. The expression levels (in terms of β-galactosidase units) varied during the phase of growth of cultures, reaching high levels during the early exponential and the stationary phase and reduced levels during entry into stationary phase. Both the levels of expression and the MICs were more elevated at lower temperatures. Cloning the gene under the control of other strong mycobacterial promoters also resulted in higher MIC values.
Conclusions: In M. smegmatis mc2155, the aminoglycoside resistance levels conferred by the AAC(2')-Id enzyme directly rely on the strength of the promoter driving transcription of the aac(2')-Id gene.
Keywords: mycobacteria , mechanisms of resistance , leaderless mRNA
Present address. Servicio de Microbiologia, Hospital Universitario de Bellvitge, Feixa Llarga s/n, 08 907-L’Hospitalet, Barcelona, Spain.
Present address. GlaxoSmithKline R&D, Diseases of the Developing World, Molecular Drug Discovery, C/ Severo Ochoa, n° 2, 28 760-Tres Cantos, Madrid, Spain.