Addiction Toxin Fst Has Unique Effects on Chromosome Segregation and Cell Division inEnterococcus faecalisandBacillussubtilis

Abstract

The Fst toxin of the Enterococcus faecalis pAD1-encoded par addiction module functions intracellularly to kill plasmid-free segregants. Previous results had shown that Fst induction results in membrane permeabilization and cessation of macromolecular synthesis, but only after 45 min. Electron micrographs of toxin-induced cells showed no obvious membrane abnormalities but did reveal defects in nucleoid segregation and cell division, begging the question of which is the primary effect of Fst. To distinguish the possibilities, division septae and nucleoids were visualized simultaneously with fluorescent vancomycin and a variety of DNA stains. Results showed that division and segregation defects occurred in some cells within 15 min after induction. At these early time points, affected cells remained resistant to membrane-impermeant DNA stains, suggesting that loss of membrane integrity is a secondary effect caused by ongoing division and/or segregation defects. Fst-resistant mutants showed greater variability in cell length and formed multiple septal rings even in the absence of Fst. Fst induction was also toxic to Bacillus subtilis. In this species, Fst induction caused only minor division abnormalities, but all cells showed a condensation of the nucleoid, suggesting that effects on the structure of the chromosomal DNA might be paramount.

Characterization of the Effects of an rpoC Mutation That Confers Resistance to the Fst Peptide Toxin-Antitoxin System Toxin

“…Glycine betaine is an environmentally available osmoprotectant (41), and the phage shock protein is induced by proton motive force dissipation (42); the other induced transporters could also be induced as an attempt by cells to maintain osmotic and ionic balances within the cell in response to some form of membrane stress. This would be consistent with the membrane localization of the Fst toxin (20) and previous results indicating that Fst-exposed cells become permeable to membrane-impermeant dyes after 45 to 60 min (24). It was previously observed that nisin and other membrane-active peptides induce a specific response by ABC transporters linked to two-component regulators (43).…”

“…Unlike another RNA-regulated peptide toxin family, the Hok/Gef toxins, overexpression of Fst/Ldr toxins does not result in the formation of "ghost cells," in which the cell center becomes translucent and the cell material appears to condense at the poles (21,22). Rather, the primary effect of overexpression of Ldr in Escherichia coli (23) and Fst in E. faecalis, Bacillus subtilis, Staphylococcus aureus, and E. coli (11,24) is the condensation of the nucleoid. In E. faecalis, nucleoid condensation is accompanied by aberrant chromosome segregation, irregular division septum placement, and loss of membrane integrity (24).…”

“…Rather, the primary effect of overexpression of Ldr in Escherichia coli (23) and Fst in E. faecalis, Bacillus subtilis, Staphylococcus aureus, and E. coli (11,24) is the condensation of the nucleoid. In E. faecalis, nucleoid condensation is accompanied by aberrant chromosome segregation, irregular division septum placement, and loss of membrane integrity (24). The primary cause of these cell morphology changes is unclear.…”

Small Toxic Proteins and the Antisense RNAs That Repress Them

“…Overexpression of the LdrD protein leads to nucleoid condensation in E. coli (31), and Fst overexpression disrupts nucleoid structure, chromosome segregation, and cell division in E. faecalis (41). For Fst, these physiological effects have been observed at lower levels of expression than those required for membrane disruption (41).…”

“…Overexpression of the LdrD protein leads to nucleoid condensation in E. coli (31), and Fst overexpression disrupts nucleoid structure, chromosome segregation, and cell division in E. faecalis (41). For Fst, these physiological effects have been observed at lower levels of expression than those required for membrane disruption (41). High levels of TisB lead to decreased DNA, RNA, and protein synthesis in vivo, but these effects are likely to be indirect consequences of membrane damage, since TisB had no effects on transcription and translation in vitro (52).…”