Untwisting of the DNA helix stimulates the endonuclease activity of Bacillus subtilis Nth at AP sites

Bacterial nucleoid associated proteins play a variety of roles in genome maintenance and dynamics. Their involvement in genome packaging, DNA replication and transcription are well documented but it is still unclear whether they play any specific roles in genome repair. We discovered that untwisting of the DNA double helix by bacterial non-specific DNA binding proteins stimulates the activity of a repair endonuclease of the Nth/MutY family involved in abasic site removal during base excision repair. The essential Bacillus subtilis primosomal gene dnaD, coding for a protein with DNA-untwisting activity, is in the same operon with nth and the promoter activity of this operon is transiently stimulated by H2O2. Consequently, dnaD mRNA levels persist high upon treatment with H2O2 compared to the reduced mRNA levels of the other essential primosomal genes dnaB and dnaI, suggesting that DnaD may play an important role in DNA repair in addition to its essential role in replication initiation. Homologous Nth repair endonucleases are found in nearly all organisms, including humans. Our data have wider implications for DNA repair as they suggest that genome associated proteins that alter the superhelicity of the DNA indirectly facilitate base excision repair mediated by repair endonucleases of the Nth/MutY family

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

The ζ toxin induces a set of protective responses and dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ~78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (~10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activityBiochemijos katedraVytauto Didžiojo universiteta

Remains of flood destroyed steel bridge, Butibum River, [Papua New Guinea, 1960?]

<p><i>xylR</i>-<i>P</i>_XylA or <i>xylR</i>-<i>P</i>_XylA Δ<i>relA</i> or <i>xylR</i>-<i>P</i>_XylAζY83C or <i>xylR</i>-<i>P</i>_XylAζY83C Δ<i>relA</i> were grown in MMS7. At ∼5×10⁷ cells/ml⁻¹ 0.5% Xyl^a (to induce ζY83C expression) or 0.5 mg ml⁻¹ Dec^a (to reduce GTP synthesis) or both^b, Xyl and Dec, were added and the culture was incubated for 120 min.</p>f<p>The presence of ζY83C toxin is indicated by yes or no.</p>g<p>Number of cells analyzed are shown in parentheses.</p>h<p>Due to poor growth of the Δ<i>relA</i> strains CFUs were measured after two days of incubation.</p>i<p>The CFUs were measured after 120 min of toxin induction by plating appropriate dilutions on LB plates. The results are the average of at least three independent experiments and are within a 10% standard error.</p

Schema of phenotypes observed upon ζ toxin expression.

<p>At time zero expression of the ζ toxin was induced. Between the 5 to 15 min interval the expression of 78 genes was altered, without apparent alteration of the cellular proteome. At indicated times intervals macromolecular biosynthesis, GTP and ATP pool was reduced, the membrane permeability altered, and a novel radiolabeled nucleotide accumulated. After 120 min ∼30% of cells became PI stained and ∼10⁻⁴ were able to form colonies after overnight incubation. In the lower line, at time 120 min after toxin expression the expression of the ε₂ antitoxin was also induced and the number of survivals and the proportion of PI stained cells estimated 120 min later (240 min).</p

Effect of Δ<i>relA</i> mutation in toxin induced PI staining and dormancy.

<p><i>xylR</i>-<i>P</i>_XylA Δ<i>relA</i> or <i>xylR</i>-<i>P</i>_XylAζY83C Δ<i>relA</i> cells were grown in MMS7.</p>a<p>0.5% Xyl was added to induce expression of the ζY83C toxin and the culture was incubated for 120 min.</p>b<p>The presence of the ζY83C toxin is indicated by yes or no.</p>c<p>Number of cells analyzed are shown in parentheses.</p>d<p>The CFUs were measured after 120 min of toxin induction by plating appropriate dilutions on LB plates. The results are the average of at least three independent experiments and are within a 10% standard error.</p

Expression of ζ toxin affects the membrane permeability.

<p>(A) <i>lacI</i>-<i>P</i>_hspζ (<i>xylR</i>-<i>P</i>_XylAε) cells were grown in two parallel vessels containing 30 ml MMS7 at 37°C up to ∼5×10⁷ cells ml⁻¹ in the presence of traces of Xyl (0.005%) and the K⁺ concentration in the medium was recorded. Then, 1 mM IPTG was added to one of the vessels and the monitoring of K⁺ concentrations in the cell suspensions was followed for 100 min (red curve). For control of the intracellular K⁺ content lysozyme (30 µg ml⁻¹) was added as well as calibration of the electrodes by 6 µmol KCl additions was performed at indicated time frames (green curve). (B) <i>lacI</i>-<i>P</i>_hspζ (<i>xylR</i>-<i>P</i>_XylAε) cells were grown in two parallel vessels in MMS7 at 37°C up to OD₅₆₀ in the presence of traces of Xyl (0.005%). Then, 1 mM IPTG was added to one of the vessels (empty circles) and OD₅₆₀ recorded. In A and B, the arrows point at the time of addition of the indicated compound.</p

Variations of free ζ toxin levels differentially affect dormancy and permeation to PI.

<p><i>lacI</i>-<i>P</i>_hspζ (<i>xylR</i>-<i>P</i>_XylAε) cells were grown in MMS7 at 37°C up to ∼5×10⁷ cells ml⁻¹ in the presence of traces of Xyl (0.005%, denoted as (+), to allow limiting ε₂ antitoxin expression to titrate basal expression of the wt ζ toxin. IPTG (1 mM) and variable amounts of Xyl (0.05, 0.1 and 0.5%) were added and the culture incubated for 120 min. Aliquots were taken and appropriate dilutions were plated in Luria-Bertani (LB) plates with the same concentration of Xyl, or analyzed under the microscope after live-dead staining. Means of four parallel experiments ±95% confidence intervals are shown.</p

Level of toxin expression and bacterial growth.

<p><i>lacI</i>-<i>P</i>_hspζ cells bearing plasmid pCB799 (<i>xylR</i>-<i>P</i>_XylAε) were grown in MMS7 containing 0.005% Xyl to allow limiting ε₂ antitoxin expression, ε⁽⁺⁾, to titrate basal expression of the wt ζ toxin.</p>a<p>Cells grown exponentially in MMS7 to ∼5×10⁷ cells ml⁻¹, a sample was collected (corresponding to 2 ml at an OD₅₆₀ of 0.4), cells lysed and subjected to immunoblot transfer for toxin detection. Cells grown exponentially in MMS7 to ∼5×10⁷ cells ml⁻¹, 0.5% Xyl^b or 1 mM IPTG^c was added, samples collected at different times.</p>d<p>The presence or the absence of induction of ζ, ζY83C or ε₂ are indicated by + or − superscript, respectively.</p>e<p>Samples were collected after 60 min of induction, equivalent amounts of cells (corresponding to 2 ml at an OD₅₆₀ of 0.4) were lysed and subjected to immunoblot transfer for toxin detection. Toxin levels are expressed as monomers/per cells.</p>f<p>Cell doubling time (in min) was measured by recording the OD₅₆₀ every 30 min until reaching early stationary phase. NA, not applicable. The results are the average of at least four independent experiments.</p