The par toxin-antitoxin system from Enterococcus faecalis plasmid pAD1 and its chromosomal homologs

Abstract

The par post-segregational killing locus present on Enterococcus faecalis plasmid pAD1 was the first Type I toxin-antitoxin system described in Gram-positive bacteria. Translation of the 33 amino acid Fst toxin, encoded on RNA I, is suppressed by a 66 nucleotide regulatory RNA, RNA II. RNA I and RNA II are transcribed convergently and interact at dispersed regions of complementarity, establishing a stable complex that accumulates in plasmid-containing cells. RNA II is slowly removed from the complex, allowing translation of RNA I in plasmid-free segregants. Intramolecular structures are also important for regulating translation of RNA I. The Fst toxin contains a putative transmembrane domain and is believed to exert its function at the bacterial cytoplasmic membrane, although its precise target and mode of action have yet to be determined. Numerous chromosomal homologs of pAD1 par have been identified in Gram-positive bacteria suggesting that this locus may play important roles in cellular function.

Keywords: antisense RNA; peptide toxins; plasmid stability; post-segregational killing; toxin-antitoxin.

Figure 1. Organization of the pAD1 par locus and the Fst toxin. Converging promoters (black arrowheads labeled P) transcribe the toxin-encoding RNA I (red shaded arrow below line) and the antitoxin RNA II (dark green arrow above line) toward a bi-directional intrinsic transcriptional terminator (converging green arrows). The RNAs are transcribed across direct repeats DRa (pink arrows) and DRb (blue arrows) at which interaction occurs, suppressing translation of the Fst_pAD1 coding sequence (dark red box on DNA and RNA). The amino acid sequence of the Fst toxin is shown using standard single letter amino acid designations. The essential, conserved hydrophobic domain is shown in bold red print. This forms part of a transmembrane domain in the recently published structure of Fst. The two blue underlined amino acids at the N-terminus must be charged to retain toxin function. The non-essential C-terminal tail is shown in green italics.

Figure 2. Secondary structures of RNA I_pAD1 and RNA II_pAD1. The specific regions of interaction between the RNAs are shaded different colors to coordinate with Figure 1 and labeled accordingly. Interaction is initiated at the U-turn motif (labeled YUNR) present in the loop of the terminator of RNA I (green shaded). This interaction is indicated by the arrow labeled A. The interaction then extends to the direct repeat sequences DRa (pink shaded) and DRb (blue shaded). This interaction is indicated by arrows labeled B and is responsible for preventing translation of Fst_pAD1, since the initiation codon (I.C.) and the ribosome binding site (SD) are sequestered by the interacting RNAs. The two structures, 5′-SL (blue box) and 5′-UH (red box) are responsible for preventing premature translation of Fst_pAD1 and RNA I_pAD1 stability, respectively.