FIG 1.
hipBST of E. coli O127 encodes a three-component toxin-antitoxin module. (A) Schematic showing a comparison of the hipBA and hipBST operons of E. coli K-12 and O127, respectively. Bent arrows pointing right indicate promoters. The hipBA operon contains two genes, hipB and hipA, while hipBSTO127 contains three genes, hipBO127, hipSO127, and hipTO127. The region of hipA between the dashed lines exhibits sequence similarity to hipSO127. The 8 amino acid residues of the P loop in HipA (150-VAGAQEKT-158) that binds phosphates of ATP are shown; the autophosphorylated S150 residue is shown in green (35). The homologous P loop and autophosphorylated serine in HipTO127 were inferred by sequence similarity. (B) Overnight cultures of E. coli MG1655 harboring pSVN1 (pBAD33::hipTO127) or the empty pBAD33 vector combined with pSVN111 (pNDM220::hipBO127), pSVN109 (pNDM220::hipSO127), pSVN110 (pNDM220::hipBSO127), or the empty low-copy-number pNDM220 vector, as indicated, were diluted to obtain the same values of OD600, centrifuged at 5,000 rpm for 5 min, washed in phosphate-buffered saline (PBS), and serially diluted before being spotted onto LB nutrient agar plates containing 0.2% glucose (to repress hipTO127), 0.2% arabinose (to induce hipTO127), or 0.2% arabinose plus 200 μM IPTG (to induce hipBO127, hipSO127, or hipBSO127). (C) The strains used in the experiment whose results are shown in panel B were grown in LB medium plus appropriate antibiotics. Overnight cultures were diluted, cells were grown exponentially for at least 3 h until the doubling time appeared constant, and at an OD600 of ≈0.3, arabinose (0.2%) was added to induce hipTO127 (red arrow). After a further 1.5 h, IPTG (200 μM) was added to induce hipSO127, hipBO127, or hipBSO127 (green arrow). (D and E) Viable counts of strains from the experiment whose results are shown in panels B and C before and after the addition of arabinose (0.2%) at an OD600 of ≈0.3 (red arrow). At each time point, cell samples (0.5 ml) were washed in PBS before a 10-times dilution series was spotted on agar plates with glucose (0.2%) to repress hipTO127 expression (D) or with glucose (0.2%) to repress hipTO127 expression and IPTG (200 μM) to induce hipBO127, hipSO127, or hipBSO127 (E). Plates were incubated for 16 h at 37°C before counting. Data points in panels C, D, and E represent mean values of results from at least three independent experiments, and error bars indicate standard deviations.