pSM19035-encoded [zeta] toxin induces stasis followed by death in a subpopulation of cells

The toxin-antitoxin operon of pSM19035 encodes three proteins: the [omega] global regulator, the [epsilon] labile antitoxin and the stable [zeta] toxin. Accumulation of [zeta] toxin free of [epsilon] antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a [zeta] variant ([zeta]Y83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free [zeta] toxin induced stasis, but this was fully reversed by expression of the [epsilon] antitoxin within a defined time window. The time window for reversion of [zeta] toxicity by expression of [epsilon] antitoxin was dependent on the initial cellular level of [zeta]. After 240 min of constitutive expression, or inducible expression of high levels of [zeta] toxin for 30 min, expression of [epsilon] failed to reverse the toxic effect exerted by [zeta] in cells growing in minimal medium. Under the latter conditions, [zeta] inhibited replication, transcription and translation and finally induced death in a fraction (~ 50 %) of the cell population. These results support the view that [zeta] interacts with its specific target and reversibly inhibits cell proliferation, but accumulation of [zeta] might lead to cell death due to pleiotropic effects.