LfrR is a repressor that regulates expression of the efflux pump LfrA in Mycobacterium smegmatis

Abstract

The lfrA gene of Mycobacterium smegmatis encodes an efflux pump which mediates resistance to different fluoroquinolones, cationic dyes, and anthracyclines. The deletion of the lfrR gene, coding for a putative repressor and localized upstream of lfrA, increased the lfrA expression. In this study, reverse transcription-PCR experiments showed that the two genes are organized as an operon, and lacZ reporter fusions were used to identify the lfrRA promoter region. The lfrRA promoter assignment was verified by mapping the transcription start site by primer extension. Furthermore, we found that some substrates of the multidrug transporter LfrA, e.g., acriflavine, ethidium bromide, and rhodamine 123, enhance lfrA expression at a detectable level of transcription. LfrR protein was purified from Escherichia coli as a fusion protein with a hexahistidine tag and found to bind specifically to a fragment 143 bp upstream of lfrR by gel shift analysis. Furthermore, acriflavine was able to cause the dissociation of the LfrR from the promoter, thus suggesting that this molecule interacts directly with LfrR, inducing lfrA expression. These results suggest that the LfrR repressor is able to bind to different compounds, which allows induction of LfrA multidrug efflux pump expression in response to these ones. Together, all data suggest that the LfrA pump is tightly regulated and that the repression and induction can be switched about a critical substrate concentration which is toxic for the cell.

FIG. 1.

Localization of the lfrRA promoter region using lacZ transcriptional fusions. A. Diagram showing the sizes and the positions of the various fragments (shown as black boxes) contained in the lacZ fusion plasmids. The white arrows indicate the lfrR and lfrA genes. B. β-Galactosidase activity of M. smegmatis cells containing the different constructs. Error bars indicate standard deviations from triplicate determinations of β-galactosidase activity.

FIG. 2.

A. Mapping of the transcriptional start point for the lfrRA operon. The position of the transcription start site was determined by primer extension with the oligonucleotide prexR (Table 1). Sequencing reactions, performed with the same oligonucleotide on a plasmid containing the entire lfrRA operon and the regions upstream and downstream of the genes, are reported in the last four lanes of the panel. The coordinates of the 5′ end are reported on the left of the panel. B. Nucleotide sequence of the lfrRA promoter region. The nucleotide sequences around the promoter region of the lfrRA genes are presented. The first 25 amino acids and the corresponding nucleotide sequence of the LfrR protein are also reported. The transcription start site is marked by an arrow. The putative −10 promoter element and the unusual −35 region at a distance of 28 bp are underlined. The “extended −10 promoter” is double underlined (see Discussion).

FIG. 3.

Effects of different LfrA substrates on lfrA expression. The expression of lfrA was analyzed by RT-PCR with primers RG391 and RG392 on RNA isolated from M. smegmatis mc²155 cultures. A. Expression of lfrA in the presence of increasing concentrations of ethidium bromide after 1 (lanes 3 to 6) and 4 h (lanes 8 to 11) of treatment. Lane 1, molecular size marker (Fermentas); also, the sizes of some bands in base pairs are provided on the left; lanes 2 and 7, no ethidium bromide; lanes 3 and 8, 0.4 μg/ml (0.05× MIC); lanes 4 and 9, 0.8 μg/ml (0.1× MIC); lanes 5 and 10, 1.6 μg/ml (0.2× MIC); lanes 6 and 11, 3.2 μg/ml (0.4× MIC); lane 12, negative control; lane 13, positive control, which is DNA amplification with primers RG391 and RG392 (expected size, 547 bp). The expression of the gyrA gene (317 bp) of M. smegmatis was determined as an internal control of all the RT-PCRs and is shown at the bottom of the panel. B. Expression of lfrA in the presence of acriflavine (lanes 2 and 3, 0.625 and 5 μg/ml [0.05× and 0.4× MIC]), doxorubicin (lanes 4 and 5, 0.2 and 1.6 μg/ml [0.05× and 0.4× MIC]), rhodamine 123 (lanes 6 and 7, 0.25 and 2 μg/ml [0.05× and 0.4× MIC]), and ciprofloxacin (lanes 10 and 11, 0.008 and 0.064 μg/ml [0.05× and 0.4× MIC]) after 2 h of treatment. Lanes 1, 8, and 9 are the same as lanes 1, 12, and 13 in panel A. The expression of the gyrA gene (317 bp) of M. smegmatis was determined as an internal control of all the RT-PCRs and is shown at the bottom of the panel. C. Expression of lfrA in M. smegmatis mc²155 wild-type (lane 2) and M. smegmatis mc²11 mutant (lane 3) strains grown in the absence of inducers. Lanes 1 and 4 are the same as lanes 1 and 13 in panel A. The expression of the gyrA gene (317 bp) of M. smegmatis was determined as an internal control of all the RT-PCRs and is shown at the bottom of the panel.

FIG. 4.

LfrR binds specifically to the DNA sequence upstream of the lfrRA operon. Lane 1, free labeled 143-bp probe; lanes 2 and 3, probe with 20 and 40 ng of LfrR, respectively; lane 4, probe with 20 ng of LfrR and a 100-fold molar excess of nonspecific Rv0191 fragment (noncompetitive probe); lanes 5 and 6, probe with 20 ng of LfrR and 100- and 50-fold molar excesses of 143-bp fragment (competitive probe), respectively; lane 7, probe with 20 ng of LfrR and 0.32 μg/ml of ciprofloxacin (2× MIC); lane 8, probe with 20 ng of LfrR and 25 μg/ml of acriflavine (2× MIC). The letter a indicates the free labeled 143-bp probe, while letters b and c indicate two different retarded complexes (see Results).