The antisense RNA of the par locus of pAD1 regulates the expression of a 33‐amino‐acid toxic peptide by an unusual mechanism

Abstract

SummaryThe par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNAregulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem±loop structure at the 5 H end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid-free cells.

Small Toxic Proteins and the Antisense RNAs That Repress Them

“…RNAI-RNAII encoded on the pAD1 plasmid of Enterococcus faecalis, the first type I toxinantitoxin pair found for a gram-positive bacterium, was identified on the basis of a postsegregational killing phenotype (54,55). For this pair, RNAI encodes the small Fst (E. faecalis plasmid-stabilizing toxin), protein while RNAII functions as the regulatory sRNA (24).…”

“…However, other interactions are required to prevent translation of fst encoded by RNAI. The RNAs have two complementary direct repeats in their 5Ј ends, and base pairing between these direct repeats is required to block ribosome access and translation both in vivo and in vitro (24,26). The 3Ј ends of the TpxA mRNA and RatA RNA overlap by approximately 75 nucleotides (49).…”

Small Untranslated RNA Antitoxin inBacillus subtilis

“…Examples of plasmid-borne TA systems that use antisense RNAs are hok/ sok (12) from plasmid R1 and flm from F plasmid (24) in E. coli and the par locus in plasmid pAD1 in Enterococcus faecalis (13). More recently, two examples of chromosomal toxin-antitoxin systems have been identified in E. coli that use an antisense RNA as an antidote.…”

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

“…However, the RNAs are transcribed in opposite directions across a pair of direct repeats, resulting in dispersed regions of complementarity more similar to chromosomally encoded RNA regulators than to traditional antisense-regulated systems (18,19,36). Binding of RNA II to RNA I suppresses the translation of a 33-amino-acid peptide designated Fst which functions as the toxin of the system (17). Overproduction of Fst results in a loss of cell viability, loss of membrane integrity, abnormalities in chromosomal segregation and cell division, and hypersensitivity to the lantibiotic nisin (39).…”