OBJECTIVES: To analyse the correlation between the expression levels of the aac(2')-Id gene from Mycobacterium smegmatis mc(2)155 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 mc(2)155 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 beta-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 mc(2)155, 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.
OBJECTIVES: To analyse the correlation between the expression levels of the aac(2')-Id gene from Mycobacterium smegmatis mc(2)155 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 mc(2)155 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 beta-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 mc(2)155, 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.
Authors: M Analise Zaunbrecher; R David Sikes; Beverly Metchock; Thomas M Shinnick; James E Posey Journal: Proc Natl Acad Sci U S A Date: 2009-11-11 Impact factor: 11.205