Three new toxin-antitoxin loci of E. coli. A. Chromosomal positions of the seven well-characterized toxin-antitoxin loci of E. coli K1: relBE, yefM yoeB, mazEF, prlF yhaV, chpSB and dinJ yafQ (black text) and the three new TA loci yafNO, higBA (ygjNM) and ygiUT (red text). All 10 loci encode mRNA interferases. B. Genetic overview of the three new TA loci, yafNO, higBA (ygjNM) and ygiUT and the flanking DNA regions. Toxin genes are coloured read, antitoxin genes are blue. The promoter regions of the TA operons are shown as a blow up below each gene pair. Transcriptional start sites were mapped (Fig. 5) and shown as broken arrows pointing rightward. The start sites of the first gene in the TA operons are also shown. Putative −10, −35 and Shine and Dalgarno (SD) sequences are indicated. Inverted repeats that are putative protein binding sites are shown as opposing arrows. The yafNO promoter has a putative LexA box. Bases that conform to the canonical LexA box are shown in bold.

Mapping of the yafNO, higBA and ygiUT promoters. Left panels: Primer extension analysis on total RNA purified from cells of either wild-type MG1655 (wt) or ΔyafN, ΔhigB, ΔygiU, Δlon, Δclp or Δlon clp. Cells were grown exponentially in LB medium and at time zero protein synthesis was inhibited with chlorampenicol (50 μg ml⁻¹). The ³²P-labelled primers, yafN PE1, ygjN PE1 and ygiU PE1 were used to map the 5′ ends of yafNO (A), higBA (B) and ygiUT (C) mRNAs respectively. The putative transcriptional start sites are marked with arrows is the text panel next to each gel. Right panels: DNA fragments carrying the expected promoter sequences were inserted upstream of lacZ in transcriptional fusions. Cells of TB28ΔyafNO (A), TB28ΔhigBA (B) or TB28ΔygiUT (C) were transformed with empty pOU254 or derivatives containing promoter fusions to lacZ and empty pCA24N or derivatives carrying the relevant antitoxins under the control of the IPTG-inducible T5-lac promoter. Cells were plated on LA containing Amp (30 μg ml⁻¹), Cml (50 μg ml⁻¹), Xgal (40 μg ml⁻¹) and IPTG (0.5 mM).

yafNO, higBA and ygiUT are bona fide toxin-antitoxin loci. A. Growth of E. coli MG1655 (wt) carrying one of the plasmids, pBAD33 (vector), pMCD3306 (pBAD::SD::yafO), pBAD3310 (pBAD::SD::higB) or pMCD3312 (pBAD::SD::ygiU). Cells were grown exponentially in LB medium at 37°C. At time zero (OD₄₅₀ ≈ 0.5), arabinose (0.2%) was added to induce transcription of the toxin genes. B–D. Cells of MG1655/pMCD3306 (pBAD::SD::yafO)/pMCD2202 (P_A1/O3/O4::SD::yafN) (B), MG1655/pMCD3310 (pBAD::SD::higB)/pMCD2205 (P_A1/O3/O4::SD::higA) (C) and MG1655/pMCD3312 (pBAD::SD::ygiU)/pMCD2207 (P_A1/O3/O4::SD::ygiT) (D) were grown exponentially in LB medium at 37°C. Transcription of the toxin genes was induced by addition of arabinose (0.2%) at time zero and cells plated on LA plates with IPTG (2 mM) () or without IPTG () to induce transcription of the antitoxin genes. The number of colony-forming units ml⁻¹ was calculated for each of the indicated time points. E. Cells of E. coli MG1655 carrying pBAD33 (vector), pMG3323 (pBAD::relE), pMCD3306 (pBAD::SD::yafO), pBAD3310 (pBAD::SD::higB) or pMCD3312 (pBAD::SD::ygiU) were grown exponentially in M9 minimal medium and the transcription of the toxins induced at time zero with arabinose (0.2%). Samples were taken at the indicated time points, and protein synthesis was measured by pulse-labelling cells for 1 min with ³⁵S-methionine and chased for 10 min with cold methionine. The pre-incubation rates were set to 100%.

Translation affects target mRNA cleavage differentially. A. Plasmid pMCD25420 produces wild type dksA mRNA whereas pMCD25421 produces a dksA mRNA with the ATG start codon changed to AAG (dksA′ mRNA). B–D. The strains MG1655Δ5ΔdksA/pMCD25420 or MG1655Δ5ΔdksA/pMCD25421 were cotransformed with the plasmids, pMCD3306, pMCD3310 and pMCD3312. Cells were grown exponentially in LB and induced with arabinose (0.2%) at time zero. Total RNA was purified and used for Northern blot analysis using an RNA probe complementary to the dksA mRNA (B) and primer extension analysis on dksA mRNA using the ³²P-labelled primer pKW71D-3#PE (C and D), where (C) is a representation of the dksA 5′ end and panel D a representation of the RNA region close to the dksA stop codon. Numbers are time points of cell sampling relative to the addition of arabinose or CML. Significant cleavage sites are indicated with black symbols and important sites in the mRNAs are indicated with arrows to the right.

Ectopic induction of yafO, higB and ygiU confers mRNA cleavage. A. Northern analysis of the lpp and ompA mRNAs using specific ³²P-labelled ribonucleoacid probes. Primer extensions on lpp (B), ompA (C) and rpoD (D) mRNAs using the ³²P-labelled primers lpp26, ompA-ctr-ccw and rpoD PE1 respectively. Total RNA was prepared from cells of MG1655Δ5 (lacks the toxin antitoxin systems relBE, yefM/yoeB, dinJ/yafQ, mazEF and chpSB) or MG1655Δ5 carrying on of the plasmids pMG3323 (pBAD::relE), pMCD3306 (pBAD::SD::yafO), pBAD3310 (pBAD::SD::higB) or pMCD3312 (pBAD::SD::ygiU) growing exponentially in LB medium at 37°C. At time zero, CML was added to MG1655Δ5 and transcription of the toxin genes from the remaining cells induced with arabinose (0.2%). Cleavage sites mediated by RelE (•), YafO (), HigB () and YgiU (⋄) are marked on the gels and in the relevant RNA sequences below each gel. The numbers above lanes are the time points of cell sampling relative to toxin induction or addition of CML.

Nutritional stresses differentially induce TA loci transcription. Cells of MG1655 were grown exponentially in M9 minimal medium at 37°C. Samples were taken before and after induction of various stress conditions at the time points indicated. Transcriptional activation of yafNO, ygiUT and, higBA (ygjNM) were analysed by reverse transcription qPCR and represented by relative fold of changes. (A) 30 μg ml⁻¹ CML; (B) 0.5 mg ml⁻¹ valine addition; (C) 1% methyl-α- D-glycopyranoside; (D) 0.4 mg ml⁻¹ serine hydroxamate; (E) 1 μg ml⁻¹ mitomycin C. Note that the different panels have different scales on their Y-axes.