JAC Advance Access published online on November 20, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm440
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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
* Corresponding author. Tel: +34-976-762420; Fax: +34-976-762604; E-mail: ainsa{at}unizar.es
Received 9 July 2007; returned 5 September 2007; revised 8 October 2007; accepted 18 October 2007
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Abstract |
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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 ß-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.
Key Words: mycobacteria , mechanisms of resistance , leaderless mRNA
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Introduction |
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Aminoglycoside-modifying enzymes (AGMEs) are one of the most common mechanisms of bacterial resistance to this family of antibiotics. N-acetyltransferases, O-phosphotransferases and O-nucleotidyltransferases may alter the aminoglycoside molecule by adding chemical groups in different positions, which prevents the binding of the modified aminoglycoside to the ribosome.1 Most genes encoding AGMEs are located on mobile elements, such as plasmids or transposons, and confer high levels of resistance to aminoglycosides (which are clinically relevant), whereas other genes are located in the chromosome and usually do not confer clinically relevant levels of aminoglycoside resistance.2–5 However, in aminoglycoside-susceptible strains of Salmonella enterica, it has been described that changes in the activity of the promoter of chromosomal genes encoding AGMEs produced an increase in the transcription of such genes that resulted in clinically relevant aminoglycoside resistance.6,7 Such activation of cryptic antibiotic resistance genes could occur in other bacterial species having chromosomally located genes encoding AGMEs normally expressed at low levels.
The mycobacteria are human pathogens causing important diseases such as tuberculosis, leprosy and serious infections in immunocompromised patients. In mycobacterial species, the presence of AGMEs activity has been detected,8–12 and chromosomal aac(2')-I or aph(3'')-I genes have been characterized.13–15 However, strains showing AGMEs activity and/or having genes encoding AGMEs usually show low-level aminoglycoside resistance. It has been speculated whether these enzymes have a primary role in aminoglycoside resistance or whether they may contribute to other housekeeping processes.9,16
In the present report, we have characterized the chromosomally located aac(2')-Id gene from Mycobacterium smegmatis [which encodes an AAC(2')-I enzyme that acetylates the 2' amino group of gentamicin, tobramycin and netilmicin and other aminoglycoside antibiotics] at the transcriptional level and have found a correlation between promoter strength levels and aminoglycoside resistance levels.
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Bacterial strains and culture conditions
M. smegmatis mc215517 and derivatives were cultured on either Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween-80 (for MIC determination, Tween-80 was replaced by 10% glycerol) or Middlebrook 7H10 agar supplemented with OADC at 37°C. Escherichia coli XL1 Blue18 and derivatives were cultured on LB broth or agar at 37°C. When required, a solution containing 1600 µg of X-Gal was added to plates. Kanamycin A (Sigma) at 20 mg/L was added where appropriate in order to maintain the plasmids.
DNA manipulation, plasmids and transformation
General molecular biology procedures were used as described previously18 or following the manufacturer instructions. E. coli XL1 and M. smegmatis mc2155 competent cells were prepared for electroporation as described previously.18,19 Cloning vectors and plasmids used in this work are described in Table 1.
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Isolation of total RNA
Cells from liquid cultures of M. smegmatis mc2155 transformed with plasmid pCL9 (Table 1) were harvested at 18 and 36 h of culture and total bacterial RNA was isolated as described previously.20 RNA samples were treated with RNase-free DNaseI (Roche) according to the manufacturer instructions, and efficiency was assessed by the absence of amplification product after a regular PCR reaction.
Identification of transcription starting points
The oligonucleotide AS-2 (5'-TCGCGGGTTTCCTGGTCCAG-3'), whose target site lies from 56 to 95 bp downstream of the start codon of the aac(2')-Id gene, was end-labelled with [
-32P]ATP using T4 polynucleotide kinase and subsequently used for primer extension studies using avian myeloblastosis virus reverse transcriptase (Promega) as described previously.20
The RNase protection assay method was carried out as described previously21 using a 250 bp RNA probe complementary to the mRNA of the aac(2')-Id gene. This RNA probe was obtained by in vitro transcription from a minigene spanning from 193 bp upstream to 32 bp downstream of the start codon of the M. smegmatis mc2155 aac(2')-Id gene that was synthesized by PCR. Once the mRNA and the RNA probe were hybridized, they were digested with an RNase cocktail.
Products of both primer extension and RNase protection assays were analysed by electrophoresis through 6% (w/v) polyacrylamide/8 M urea sequencing gels, along with DNA sequencing reactions for size determinations. Radioactive products were detected by autoradiography at –70°C using an intensifying screen.
Bacterial strains were cultured in a shaking incubator. At each time point, aliquots were removed in quadruplicate and processed for determining the level of ß-galactosidase activity using the ONPG (o-nitrophenyl-ß-D-galactoside) hydrolysis assay,18 and the number of Miller units was calculated as previously described.18 The full experiment (bacterial culture and ß-galactosidase determination) was repeated three times to ensure reproducibility.
The aminoglycoside resistance levels of M. smegmatis strains containing plasmids carrying the aac(2')-Id gene were tested by antibiotic microdilution assays using resazurin as indicator of cell growth.22 Although the kanamycin resistance proteins produced by the cloning vectors pSUM36, pMV261 and pMIP12 have not been described to confer cross-resistance with gentamicin, tobramycin or netilmicin, we included cells harbouring the cloning vectors as negative controls. Gentamicin, tobramycin and netilmicin were purchased from Sigma.
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Characterization of the promoter and expression levels of the acc(2')-Id gene
We first constructed a fusion of the promoter region and 442 nt of the 5'-portion of the aac(2')-Id gene in frame with the promoterless lacZ gene, using vector pJEM13,23 resulting in plasmid pVMS20. This plasmid was introduced in M. smegmatis mc2155, giving pale blue colonies on plates containing X-Gal, whereas the introduction of the vector pJEM13 produced no change in the colour of the colonies (data not shown). The levels of ß-galactosidase produced by M. smegmatis mc2155 cells containing either pVMS20 or pJEM13 (as a negative control) fluctuated along the growth curve as depicted in Figure 1, following the same pattern at 37°C (Figure 1a) and at 32°C (Figure 1b), although at the latter temperature the levels of ß-galactosidase were between two and four times higher than those obtained at 37°C at any time point.
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Detection and analysis of the aac(2')-Id mRNA
We isolated total RNA from cultures of M. smegmatis mc2155 transformed with plasmid pCL9 which carries the aac(2')-Id gene.14 Using primer extension analysis, we detected two main signals corresponding to putative sites of initiation of transcription located at 38 and 1 nt upstream of the GTG start codon of the aac(2')-Id gene (Figure 2a); both putative promoters have been corroborated by ribonuclease protection assay (Figure 2b) and have been designated P1 and P2, respectively. The latter promoter, which has stronger activity as determined by primer-extension analysis (Figure 2a), was predicted with a high probability by the algorithm used by the Neural Network Promoter Prediction web server (http://www.fruitfly.org/seq_tools/promoter.html).
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At the P1 promoter, starting transcription 38 nt upstream of the GTG, the hexanucleotide TGCACT is rather similar to the consensus TTGACn of –35 sequences proposed for group A of mycobacterial promoters (those having –10 and –35 putative hexamers similar to the E. coli sigma-70-dependent promoters),24 but the sequence GGCGACC around the –10 nt is not similar to the suggested consensus (TAnnnT) for this group of promoters. Concerning the P2 promoter, which drives transcription from just 1 nt upstream of the start codon thus producing a leaderless mRNA, the sequence TGACG is also similar to the proposed –35 consensus region of mycobacterial promoters.24 Besides, the –10 region of P2 was identified in the sequence TACAAC, which is identical in five of six positions to the proposed –10 region of the M. smegmatis sigA gene and S69 promoters (TACAAT).24 This sequence is preceded by the TGG trinucleotide constituting an extended –10 motif,25 a characteristic shared with putative promoters of other aac(2')-I genes detected in Mycobacterium fortuitum,13 Mycobacterium tuberculosis (http://genolist.pasteur.fr/TubercuList/), Mycobacterium leprae (http://genolist.pasteur.fr/Leproma/) and M. ulcerans (http://genolist.pasteur.fr/BuruList/).
Deletions in the 5' region of the aac(2')-Id gene confirmed the P1 and P2 promoters
In order to confirm in vivo the activity of the P1 and P2 promoters, we analysed the level of aminoglycoside resistance of M. smegmatis mc2155 containing a series of plasmids with deletions in the 5' region of the aac(2')-Id gene (Table 2). Plasmids pCL42, pCL47 and pCL44 containing both P1 and P2 promoters in the 385, 183 and 104 nt upstream of the start codon, respectively, conferred resistance to 5 mg/L of either gentamicin, tobramycin or netilmicin to M. smegmatis mc2155. In plasmid pCL45, the weak P1 promoter has been removed so that only the strong P2 promoter has been left in the 40 nt upstream of the start codon of the aac(2')-Id gene; this plasmid conferred resistance to 4 mg/L gentamicin in M. smegmatis mc2155. We consider that the moderate decrease in the level of resistance to gentamicin conferred by this plasmid is a consequence of the loss of the P1 promoter. In the same experiment, strain containing plasmid pCL46, in which the deletion removed beyond the start codon, showed an MIC of gentamicin, tobramycin or netilmicin of 2 mg/L, comparable to the MICs for either untransformed cells or cells transformed with the cloning vector pSUM36.
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The results confirm that the main promoter of the aac(2')-Id gene lies in the 40 nt left in plasmid pCL45 and would produce a leaderless mRNA consistent with the results of primer extension.
Finally, we checked whether the MIC of gentamicin, tobramycin and netilmicin changes when the aac(2')-Id gene was located under the control of strong promoters. For this, we constructed plasmids pALQ12 and pALQ13, in which the transcription of the aac(2')-Id gene is controlled by the promoter of the hsp60 gene from M. tuberculosis, and by the promoter pBlaf* from M. fortuitum, respectively, two of the strongest promoters described to date in mycobacteria (Table 1).26–28
Cells containing pALQ12 showed an MIC of gentamicin of 18 mg/L, and those containing pALQ13 showed an MIC of 14 mg/L (Table 2). The MICs of netilmicin also increased to 12 and 10 mg/L in M. smegmatis mc2155 containing pALQ12 and pALQ13, respectively. In contrast, overexpressing the aac(2')-Id gene had a less pronounced effect on the MICs of tobramycin (Table 2). In all these cases, the MICs were well above the MICs for M. smegmatis mc2155 cells containing the cloning vectors pMV261 or pMIP12.
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Aminoglycoside acetyltransferases are well-known antibiotic resistance enzymes in many bacteria. AAC(2')-I enzymes have been characterized only in the Gram-negative Providencia stuartii and in species of the genus Mycobacterium, being encoded by genes located in the chromosome.13,14,29
We have studied the expression of the aac(2')-Id gene from M. smegmatis mc2155, and its correlation with aminoglycoside resistance levels. We provide evidence that this gene is expressed at a low level, because its mRNA is detected at low level (Figure 2), and the levels of ß-galactosidase in gene fusion studies are also low (Figure 1). In accordance with the low level of expression, the resistance levels conferred by the AAC(2')-Id enzyme are also low (Table 2). However, when we expressed the gene under the control of heterologous strong promoters, the resistance levels increased (Table 2). It is conceivable that naturally occurring mutations could also increase both gene expression and resistance levels. For all these reasons, aac(2')-Id should be considered as cryptic resistance gene in M. smegmatis mc2155.
Since the aac(2')-Ia gene of P. stuartii also displays the features of a cryptic resistance gene,29 then other aac(2')-I genes, which are present in pathogenic species such as M. tuberculosis, M. fortuitum and others,13,14 could behave in the same manner. In fact, M. tuberculosis could have many other cryptic resistance genes (possibly as yet unknown), which normally would be expressed at low level and hence would not confer clinically relevant drug resistance, but whose expression could be increased by mutations, then contributing to the development of higher levels of drug resistance.
Our study revealed that the aac(2')-Id gene is expressed from two promoters, namely P1 and P2, each producing a canonical and a leaderless mRNA, respectively. None of them resemble that of the P. stuartii aac(2')-Ia gene,29 but this can be attributed to the high GC content of mycobacterial genomes, which in general makes their promoters quite different from those characterized in other organisms. In mycobacteria, TGN sequences are associated with –10 boxes of promoters constituting the so-called ‘extended –10 motifs’. Such promoters are considered to be stronger than those lacking this sequence.25 The –10 box of the P2 promoter of aac(2')-Id contains a TGG trinucleotide, and in fact, we detected a higher proportion of leaderless aac(2')-Id mRNA (produced from P2 promoter) than canonical mRNA produced from P1 (Figure 2a).
Leaderless mRNAs are expressed more actively at lower temperatures.30 Since P2, the stronger promoter of the aac(2')-Id gene, produces a leaderless mRNA, we explored the effect of temperature on gene expression. When the M. smegmatis cultures were grown at 32°C, we detected higher levels of ß-galactosidase than those from cultures grown at 37°C.
Although not common, other leaderless mRNAs have been described in prokaryotes.31,32 Within the actinomycetes, in the genus Mycobacterium, the blaF gene encoding the ß-lactamase from M. fortuitum,27 the purC gene from M. tuberculosis33 and one of the promoters of the oxyR gene from M. leprae34 also produce leaderless mRNA, and in the genus Streptomyces, leaderless mRNAs are often associated with the production of proteins that confer resistance to antibiotics that, like aminoglycosides, target the ribosome.31
Our results on the abundance of aac(2')-Id mRNA and the levels of ß-galactosidase activity suggest that the expression of this gene must be controlled in order to provide appropriate levels of the protein for each phase of the growth curve or in response to the changing environmental conditions. The total amount of specific aac(2')-Id mRNA at the two time points at which RNA has been analysed (Figure 2a) was followed by similar variations in the levels of ß-galactosidase along the growth curve (Figure 1), being both high in the middle of the exponential phase of growth (in fact, the levels of ß-galactosidase reached a peak at this time point) but dropping to very low levels transiently, when the culture is about to enter into the stationary phase. Once in stationary phase, the levels of ß-galactosidase increased again and were kept rather constant during this phase of growth. In contrast, the abundance of the aac(2')-Ia mRNA in P. stuartii cultures reaches a peak when the culture enters into the stationary phase.29 This can be attributed to differences in gene regulation in both organisms. In fact, in P. stuartii, a complex regulatory network controlling the expression of the aac(2')-I gene has been characterized,29 whereas in mycobacteria, the regulation of the expression of the aac(2')-I gene has not been explored yet. It could be tempting to consider some relationships between modulating the requirement for the aac(2')-Id gene and modulating the requirement for the protein synthesis by the mycobacteria in certain moments such as when the bacteria enter the stationary phase, a moment at which, for yet unknown reasons, the levels of this enzyme must be tightly reduced transiently. Further experiments are under progress to give insights into this phenomenon and to assess a role to the AAC(2')-Id enzyme in M. smegmatis mc2155.
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This work was funded by European Union grants QLK2-CT-2000-017610 (C. M.) and ICA4-CT-2002-10 063 (M. J. G.) and Spanish Ministry of Science and Education grants BIO2002-01 297 and BIO2005-01 810 (J. A. A.). M. J. R. received a CAM fellowship 08.2/0009/2001, and A. L. is a recipient of a Gobierno de Aragón fellowship.
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None to declare.
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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.
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We acknowledge Alberto Cebollada for enthusiastic support in elaborating Figures 1 and 2 and Eamonn Gormley for critical reading of the manuscript.
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