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Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

Erental A, Sharon I, Engelberg-Kulka H - PLoS Biol. (2012)

Bottom Line: The mazEF-mediated pathway reduces recA mRNA levels.Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations.In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

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The effect of DNA damage on membrane depolarization is recA-dependent.(A–D) E. coli (A) MC4100relA+ΔmazEF, (B) MC4100relA+ΔrecA, (C) MC4100relA+ΔmazEFΔrecA, and (D) MC4100relA+ΔyfiDΔrecA cells were grown, treated with NA (100 µg/ml), and stained with DiBAC4 as in Figure 1. Samples treated with NA are shown in red. Control samples without treatment are shown in black. (E and F) Complementation assay with plasmid pQE32-recA. E. coli strains (E) MC4100relA+ΔmazEFΔrecA and (F) MC4100relA+ΔyfiDΔrecA harboring pQE32-recA were grown and treated with NA (100 µg/ml) and in the presence (red) or absence (black) of 1 mM IPTG. The cultures were stained with DiBAC4, and the fluorescence intensity (FI) was determined by FACS as described in Figure 1. (G–J) The fluorescence intensity of DiBAC4 in cells of MC4100relA+ΔmazEF and MC4100relA+ΔrecA that were treated with NA was also determined by confocal microscopy. (K–N) Confocal microscopy of the cells treated with NA as in (G–J), but stained with the Live/Dead Kit. All data are representative of three independent experiments.
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pbio-1001281-g004: The effect of DNA damage on membrane depolarization is recA-dependent.(A–D) E. coli (A) MC4100relA+ΔmazEF, (B) MC4100relA+ΔrecA, (C) MC4100relA+ΔmazEFΔrecA, and (D) MC4100relA+ΔyfiDΔrecA cells were grown, treated with NA (100 µg/ml), and stained with DiBAC4 as in Figure 1. Samples treated with NA are shown in red. Control samples without treatment are shown in black. (E and F) Complementation assay with plasmid pQE32-recA. E. coli strains (E) MC4100relA+ΔmazEFΔrecA and (F) MC4100relA+ΔyfiDΔrecA harboring pQE32-recA were grown and treated with NA (100 µg/ml) and in the presence (red) or absence (black) of 1 mM IPTG. The cultures were stained with DiBAC4, and the fluorescence intensity (FI) was determined by FACS as described in Figure 1. (G–J) The fluorescence intensity of DiBAC4 in cells of MC4100relA+ΔmazEF and MC4100relA+ΔrecA that were treated with NA was also determined by confocal microscopy. (K–N) Confocal microscopy of the cells treated with NA as in (G–J), but stained with the Live/Dead Kit. All data are representative of three independent experiments.

Mentions: Since both membrane depolarization and DNA fragmentation were caused in the ΔmazEF derivative only by DNA damage, we suspected that ALD is connected to recA, a system well known to be involved in the SOS response [40]–[43]. Deleting recA from the WT MC4100relA+ strain did not permit membrane depolarization, as manifested by DiBAC4 staining (Figures 4B and S3J). However, deleting recA from MC4100relA+ΔmazEF prevented membrane depolarization (Figure 4C). In addition, recently, we found that yfiD specified for a death protein that acted the farthest downstream from mazEF (data not shown). As in the case of ΔmazEF, deleting recA from the derivative strain ΔyfiD also prevented membrane depolarization (Figure 4D). Moreover, when these ΔrecA strains harbored plasmid pQE32-recA, which bears an isopropyl-β-D-thio-galactopyranoside (IPTG)–inducible recA gene, and in the presence of IPTG, the membrane became depolarized (Figure 4E and 4F), suggesting that ALD was mediated through recA. On the other hand, recA is not involved in the mazEF-mediated death pathway; deleting recA does not prevent the operation of mazEF-mediated death. The addition of NA to the MC4100relA+ΔrecA strain still permitted its staining by PI, included in the Live/Dead Kit (Figures 4N and S3P). On the other hand, DiBAC4 staining (characteristic for ALD) is prevented in this strain (Figures 4J and S3J). These results also shed light on another aspect of DiBAC4 staining. Although ΔmazEF cells induced by DNA damage are longer than their WT counterparts (Figures 1D, 1F, S3B, S3D, S3F, and S3H), this is not the reason for the uptake of DiBAC4 by these cells. This is demonstrated by the fact that ΔrecA cells, which appear to be even longer than ΔmazEF cells, are not stained by DiBAC4 but rather PI (red) (see Figures 4G–4N, S3I–S3J, S3O, and S3P).


Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

Erental A, Sharon I, Engelberg-Kulka H - PLoS Biol. (2012)

The effect of DNA damage on membrane depolarization is recA-dependent.(A–D) E. coli (A) MC4100relA+ΔmazEF, (B) MC4100relA+ΔrecA, (C) MC4100relA+ΔmazEFΔrecA, and (D) MC4100relA+ΔyfiDΔrecA cells were grown, treated with NA (100 µg/ml), and stained with DiBAC4 as in Figure 1. Samples treated with NA are shown in red. Control samples without treatment are shown in black. (E and F) Complementation assay with plasmid pQE32-recA. E. coli strains (E) MC4100relA+ΔmazEFΔrecA and (F) MC4100relA+ΔyfiDΔrecA harboring pQE32-recA were grown and treated with NA (100 µg/ml) and in the presence (red) or absence (black) of 1 mM IPTG. The cultures were stained with DiBAC4, and the fluorescence intensity (FI) was determined by FACS as described in Figure 1. (G–J) The fluorescence intensity of DiBAC4 in cells of MC4100relA+ΔmazEF and MC4100relA+ΔrecA that were treated with NA was also determined by confocal microscopy. (K–N) Confocal microscopy of the cells treated with NA as in (G–J), but stained with the Live/Dead Kit. All data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295820&req=5

pbio-1001281-g004: The effect of DNA damage on membrane depolarization is recA-dependent.(A–D) E. coli (A) MC4100relA+ΔmazEF, (B) MC4100relA+ΔrecA, (C) MC4100relA+ΔmazEFΔrecA, and (D) MC4100relA+ΔyfiDΔrecA cells were grown, treated with NA (100 µg/ml), and stained with DiBAC4 as in Figure 1. Samples treated with NA are shown in red. Control samples without treatment are shown in black. (E and F) Complementation assay with plasmid pQE32-recA. E. coli strains (E) MC4100relA+ΔmazEFΔrecA and (F) MC4100relA+ΔyfiDΔrecA harboring pQE32-recA were grown and treated with NA (100 µg/ml) and in the presence (red) or absence (black) of 1 mM IPTG. The cultures were stained with DiBAC4, and the fluorescence intensity (FI) was determined by FACS as described in Figure 1. (G–J) The fluorescence intensity of DiBAC4 in cells of MC4100relA+ΔmazEF and MC4100relA+ΔrecA that were treated with NA was also determined by confocal microscopy. (K–N) Confocal microscopy of the cells treated with NA as in (G–J), but stained with the Live/Dead Kit. All data are representative of three independent experiments.
Mentions: Since both membrane depolarization and DNA fragmentation were caused in the ΔmazEF derivative only by DNA damage, we suspected that ALD is connected to recA, a system well known to be involved in the SOS response [40]–[43]. Deleting recA from the WT MC4100relA+ strain did not permit membrane depolarization, as manifested by DiBAC4 staining (Figures 4B and S3J). However, deleting recA from MC4100relA+ΔmazEF prevented membrane depolarization (Figure 4C). In addition, recently, we found that yfiD specified for a death protein that acted the farthest downstream from mazEF (data not shown). As in the case of ΔmazEF, deleting recA from the derivative strain ΔyfiD also prevented membrane depolarization (Figure 4D). Moreover, when these ΔrecA strains harbored plasmid pQE32-recA, which bears an isopropyl-β-D-thio-galactopyranoside (IPTG)–inducible recA gene, and in the presence of IPTG, the membrane became depolarized (Figure 4E and 4F), suggesting that ALD was mediated through recA. On the other hand, recA is not involved in the mazEF-mediated death pathway; deleting recA does not prevent the operation of mazEF-mediated death. The addition of NA to the MC4100relA+ΔrecA strain still permitted its staining by PI, included in the Live/Dead Kit (Figures 4N and S3P). On the other hand, DiBAC4 staining (characteristic for ALD) is prevented in this strain (Figures 4J and S3J). These results also shed light on another aspect of DiBAC4 staining. Although ΔmazEF cells induced by DNA damage are longer than their WT counterparts (Figures 1D, 1F, S3B, S3D, S3F, and S3H), this is not the reason for the uptake of DiBAC4 by these cells. This is demonstrated by the fact that ΔrecA cells, which appear to be even longer than ΔmazEF cells, are not stained by DiBAC4 but rather PI (red) (see Figures 4G–4N, S3I–S3J, S3O, and S3P).

Bottom Line: The mazEF-mediated pathway reduces recA mRNA levels.Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations.In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

Show MeSH
Related in: MedlinePlus