LVIS553 transcriptional regulator specifically recognizes novobiocin as an effector molecule

Abstract

In this study we aimed to identify small molecules with high affinity involved in the allosteric regulation of LVIS553, a MarR member from Lactobacillus brevis ATCC367. Using high throughput screening, novobiocin was found to specifically bind LVIS553 with a K(D) = 33.8 +/- 2.9 microM consistent with a biologically relevant ligand. Structure guided site-directed mutagenesis identified Lys(9) as a key residue in novobiocin recognition. The results found in vitro were correlated in vivo. An increased tolerance to the antibiotic was observed when LVIS553 and the downstream putative transport protein LVIS552 were either expressed in a low copy plasmid in L. brevis or as a single copy chromosomal insertion in Bacillus subtilis. We provide evidence that LVIS553 is involved in the specific regulation of a new mechanism of tolerance to novobiocin.

FIGURE 1.

Identification of the LVIS553 binding site. The genomic environment of LVIS553 was extracted and analyzed to identify putative binding regions. Two membrane proteins were encoded upstream and downstream of LVIS553. Different primer combinations (Table 1) were used to determine the smallest DNA binding region for LVIS553. EMSA results are summarized in the inset. +, positive binding of LVIS553 using 10 nm pure protein; −, negative binding using up to 500 nm purified LVIS553 protein.

FIGURE 2.

Analysis of the interaction between LVIS553 with the P_LVIS553 promoter. A, DNase I footprinting. The electropherogram shows a fragment of the digested probe in the absence (black) or presence (white) of LVIS553 highlighting the protected region. The reaction mixture was treated as described under “Experimental Procedures” using as probe the primers shown under Table 1. The nucleotide sequence protected by LVIS553 is shown in the bottom of panel A and boxed in B. B, analysis of P_LVIS553 promoter. Predicted Shine-Dalgarno sequence and −10 and −35 of the P_LVIS553 are underlined. The protected regions of both plus and minus strands are indicated in a circled box. IR, inverted repeats; MR, mirror region. C, competition EMSA. Labeled P_LVIS553 promoter with 10 nm LVIS553 were mixed with increasing concentrations of unlabeled double-stranded FP-553 (30 bp-sequence identified by DNase I footprinting).

FIGURE 3.

Effect of different small molecules on LVIS553 binding to P_LVIS553. EMSA results using 2.5 nm biotin-labeled P_LVIS553 and 10 nm LVIS553 with different inducer molecules (A) or with increasing concentrations of novobiocin (B) or coumermycin A1 (C). No protein was added to the first lane. The full binding conditions are described under “Experimental Procedures.” In A, lane 1 shows the migration of the target DNA fragment; lanes 2–7, LVIS553 at 10 nm. Carbamezepine (lane 3), canrenoic acid (lane 4), diazoxide (lane 5), pindolol (lane 6), zomepirac (lane 7), and novobiocin (lane 8) were added at a concentration of 100 μm.

FIGURE 4.

Heterologous expression of LVIS552 and LVIS553 in L. brevis and B. subtilis. A, L. brevis cells were grown with increasing concentrations of novobiocin in the presence (closed squares) or absence (closed circles) of LVIS552-LVIS553 cloned in pRV610. B, growth of B. subtilis with a chromosomal insertion of LVIS552-LVIS553 in the sacA locus (closed squares), empty controls (kanamycin cassette in the sacA locus, closed circles), and LVIS552-LVIS553 (K9A) mutant (open squares). Growth was expressed as a percentage of the OD of cells grown without novobiocin.

FIGURE 5.

Isothermal titration calorimetric data for the binding of novobiocin to LVIS553. Heat changes (upper panel) and integrated peak areas (lower panel) for the injection of a series of 4-μl aliquots of 1 mm ligand in a solution of 40 μm protein. Protein solutions at 40 μm in 10 mm HEPES, pH 7.5, and 500 mm NaCl were titrated with 1 mm solution of effector. Experiments were carried out at 30 °C.

FIGURE 6.

Close view (6-Å radius) of the SAL1 binding site of MTH313 (PDB code 3BPX) (2) aligned with the predicted model for LVIS553. A, model of the LVIS553 structure (in green) constructed using MTH313 (in blue) as a template. B, salicylate from MTH313 (in red) is displayed.

FIGURE 7.

Distinctive functions of Lys⁹ and Arg¹⁶ residues identified by EMSA. A, DNA binding properties of LVIS553 substitution mutants. WT, wild type protein. B, effect of novobiocin on the stability of LVIS553-K9A·P_LVIS553 complex.