Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression

Abstract

Toxin-antitoxin (TA) complexes function in programmed cell death or stress response mechanisms in bacteria. The YefM-YoeB TA complex of Escherichia coli consists of YoeB toxin that is counteracted by YefM antitoxin. When liberated from the complex, YoeB acts as an endoribonuclease, preferentially cleaving 3' of purine nucleotides. Here we demonstrate that yefM-yoeB is transcriptionally autoregulated. YefM, a dimeric protein with extensive secondary structure revealed by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, is the primary repressor, whereas YoeB is a repression enhancer. The operator site 5' of yefM-yoeB comprises adjacent long and short palindromes with core 5'-TGTACA-3' motifs. YefM binds the long palindrome, followed sequentially by short palindrome recognition. In contrast, the repressor-corepressor complex recognizes both motifs more avidly, impyling that YefM within the complex has an enhanced DNA-binding affinity compared to free YefM. Operator interaction by YefM and YefM-YoeB is accompanied by structural transitions in the proteins. Paired 5'-TGTACA-3' motifs are common in yefM-yoeB regulatory regions in diverse genomes suggesting that interaction of YefM-YoeB with these motifs is a conserved mechanism of operon autoregulation. Artificial perturbation of transcriptional autorepression could elicit inappropriate YoeB toxin production and induction of bacterial cell suicide, a potentially novel antibacterial strategy.

Figure 1

Autoregulation of the yefM-yoeB module. (A) Nucleotide sequence of the region 5′ of yefM-yoeB. The transcription start-site mapped by primer extension is marked as +1. Hexameric promoter motifs are boxed and the yefM start codon is in bold. The 5′ end of the yefM-yoeB transcript previously determined by RNA sequencing (41,42) is indicated by the filled circle. Long (L) and short (S) palindromes recognized by YefM-YoeB are denoted by inverted arrows. (B) Primer extension analysis of the yefM-yoeB module. Total RNA from E.coli DH5α harbouring a plasmid possessing the yefM-yoeB operon was subjected to primer extension analysis (E) using a 5′-biotinylated primer that anneals within flanking vector sequences. Reactions were performed and analysed as outlined in Materials and Methods, and electrophoresed on a denaturing 6% polyacrylamide gel in parallel with nucleotide sequencing reactions (A, C, G, T) carried out with the same biotinylated primer. The major product from the primer extension is marked as +1. (C) Autoregulation of yefM-yoeB expression by YefM and YefM-YoeB. A transcriptional fusion of the yefM-yoeB regulatory region to the lacZYA operon in plasmid pRS415 (pRSyy_wt) was transformed into E.coli SC301467, which is deleted of five chromosomal TA cassettes including yefM-yoeB, and β-galactosidase levels determined without YefM, and with YefM (pBADyefM) or YefM-YoeB (pBADyefMyoeB) supplied in trans from the pBAD33 arabinose-inducible vector (filled columns). Similar experiments were performed with a transcriptional fusion in which the S palindrome was mutated (pRSyy_Smut) (open columns).

Figure 2

YefM and YefM-YoeB binding to the yefM-yoeB promoter-operator region. (A) A 99 bp double-stranded oligonucleotide (0.1 nM) that was 5′ biotinylated on one strand and that included the yefM translation start codon and 74 bp upstream was subjected to EMSA using increasing amounts of native YefM. YefM concentrations used (left to right) (μM): 0, 0.032, 0.064, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0. Reactions were processed as outlined in Materials and Methods. Open and filled arrows denote positions of unbound oligonucleotide and YefM–DNA complexes, respectively. (B) EMSA of the same biotinylated oligonucleotide as in (A) using increasing amounts of YefM–YoeB–His₆. Protein concentrations used (left to right) (μM): 0, 0.003, 0.006, 0.012, 0.024, 0.048, 0.1, 0.2, 0.4 and 0.8. Open and filled arrows denote positions of unbound oligonucleotide and YefM–YoeB–His₆–DNA complexes, respectively. (C) EMSA of the same biotinylated oligonucleotide as in (A) with YefM–YoeB reconstituted from individual native proteins. The first three lanes contained no protein, YefM only (1 μM) or YoeB only (1 μM). The following lanes contained YoeB (1 μM) and increasing concentrations of YefM (μM): 0.125, 0.250, 0.5, 1.0, 2.0, 4.0 and 8.0. Open and filled arrows denote positions of unbound oligonucleotide and YefM–YoeB–DNA complexes, respectively. (D) Top, nucleotide sequence of the yefM-yoeB promoter–operator region with substitution mutations (stars) that disrupt the S palindrome. Bottom, EMSA of a 99 bp oligonucleotide substrate harbouring the S palindrome mutations without added protein, with native YefM (8 μM) and with YefM–YoeB–His₆ (6 μM).

Figure 3

DNase I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His₆ concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His₆. A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His₆ is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.

Figure 4

CD analysis of the YefM and YefM–YoeB–His₆ proteins in the absence and presence of DNA. (A) Far UV CD spectrum of YefM alone (10 μM), and in the presence of 2 μM 99 bp oligonucleotides with the yefM-yoeB promoter–operator region, the same region but with mutations in the S palindrome (Figure 2D), and without the promoter–operator sequences. Spectra of complexes are difference spectra as any contribution of the oligonucleotides to the spectra is subtracted from the spectrum of the complex. (B) Far UV CD spectrum of YefM–YoeB–His₆ alone (5 μM), and in the presence of 99 bp oligonucleotides with the yefM-yoeB promoter–operator region, the same region but with mutations in the S palindrome (Figure 2D), and without the promoter–operator sequences. (C) Thermal denaturation of native YefM (10 μM) monitored by CD at 222 nm. Denaturation of YefM was followed from 5–80°C (black line). YefM renaturation was subsequently analysed (red line). (D) Far UV spectra of native YefM (20 μM) before (black line) and after (red line) thermal denaturation.

Figure 5

The 1D proton spectrum of YefM (150 μM) in 50 mM Tris (pH 8.5), 150 mM NaCl at 30°C. Regions of resolved methyl and amide resonances are highlighted.

Figure 6

A section of the 600 MHz 2D ¹H-¹H NOESY spectrum (τ_m 100 ms) of YefM (150 μM) in 50 mM Tris (pH 8.5), 150 mM NaCl at 30°C. Regions containing cross peaks from regular secondary and tertiary structures are shown: (A) NH-NH, (B) NH-αH and (C) aromatic/NH-methyl protons.

Figure 7

Analysis of the solution oligomeric state of YefM. (A) Timecourse (minutes) of YefM cross-linking performed at 22°C in the presence of DMP (10 mM). Samples were electrophoresed on a 15% SDS–polyacrylamide gel. Different species formed are indicated by arrows (right) and molecular marker weights are expressed in kDa (left). (B) Timecourse (minutes) of YefM–YoeB–His₆ cross-linking performed at 22°C in the presence of DMP (10 mM). Samples were electrophoresed on a 15% SDS–polyacrylamide gel. Different species formed are indicated by arrows (right) and molecular marker weights are expressed in kDa (left). (C) Molar mass distribution of YefM. Bottom, the solid line is the trace from the refractive index indicator. Peak area selected for analysis is between the two dashed lines. Dots within the peak area are the weight average molecular weights for each slice, i.e. measured every second. Top, hydrodynamic radius (nm) versus volume (ml) across the peak.

Figure 8

Paired 5′-TGTACA-3′ motifs in the promoter regions of yefM-yoeB operons from diverse bacteria. Sequences are aligned at the ATG start codons of the yefM homologues. Paired 5′-TGTACA-3′ motifs, or motifs that possess no more than one mismatch, and that are separated by centre-to-centre distances of 12 bp are boxed. In one case, the spacing is 18 bp.

Figure 9

GlobPlot prediction (40) of intrinsic disorder in YefM. Descending regions correspond to putative domains, whereas the ascending region between residues ∼70–80 is predicted to be disordered.