The baeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate

Abstract

Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible (Delta)acrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the (Delta)acrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.

FIG. 1.

Organization of the yegMNOB baeSR locus in wild-type E. coli (A) and in the strains AG100A ΔbaeSR::cat (B), AG100A ΔyegMNOB::cat (C), AG100A ΔyegMNOB::cat (D), AG100A ΔyegOB::cat (E), AG100A ΔyegB::cat (F), and AG100A ΔyegN ΔyegB::cat (G). Note that we propose to rename the locus yegMNOB as mdtABCD (see Results).

FIG. 2.

Construction of the strain AG100A ΔyegN ΔyegB::cat. (1) Genomic DNA from the strain AG100A ΔyegOB::cat is used as a template in a PCR with a hybrid primer whose sequence at the 5′ end corresponds to the last 45 nucleotides of yegM followed by nucleotides 2 to 21 of yegO. (2) The product of the PCR described above is introduced into strain AG100A ΔyegNOB expressing Red recombinase. Two possible recombination events, labeled A and B, can lead to insertion of the cat gene into the genome. (3) Chloramphenicol-resistant clones are screened by PCR to select mutants in which recombination occurred according to mechanism A, resulting in the insertion of the yegO gene next to the yegM gene. Note that we propose to rename the locus yegMNOB as mdtABCD (see Results).

FIG. 3.

Novobiocin accumulation is reduced in acrAB cells overexpressing BaeR. Mid-exponential-phase cells were induced with 1 mM IPTG for 1.5 h, concentrated 8 times in a buffer containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM MgSO₄, and 0.2% glucose, and exposed to 10 μg of novobiocin/ml for 20 min. Cells were disrupted, and novobiocin levels were determined by HPLC. Samples contained 940 μg of total protein in a 1-ml volume. A novobiocin standard (50 ng) was used for reference.

FIG. 4.

Effect of BaeR on the activity of different reporter constructs. LMG194 cells combining the indicated reporter plasmids with either pBAD/Myc-His A (bars labeled A) or pBADBaeR (bars labeled B) were grown in LB medium at 37°C to the mid-exponential phase. l-Arabinose (0.2%) was added to one-half of each culture, and β-galactosidase activity was measured 3 h later.

FIG. 5.

Electrophoretic mobility shift assay of the interaction of BaeR-His₆ with the acrA (lanes 1 to 5) and yegM (mdtA) (lanes 6 to 11) promoter regions. ³²P-labeled DNA fragments were present at 1 nM in reaction mixtures that contained no BaeR-His₆ (lanes 1 and 6), 16 nM BaeR-His₆ (lane 11), 49 nM BaeR-His₆ (lanes 5 and 10), 0.15 μM BaeR-His₆ (lanes 4 and 9), 0.44 μM BaeR-His₆ (lanes 3 and 8), and 1.32 μM BaeR-His₆ (lanes 2 and 7). A 250-fold excess (50 μg/ml) of unlabeled competitor [poly(dI-dC)] was included in all reaction mixtures. Arrows indicate the positions of migration of discrete complexes formed between BaeR-His₆ and the probe containing the yegM (mdtA) promoter region.