No abstract

The par stability determinant of Enterococcus faecalis plasmid pAD1 is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, that function as the toxin (Fst)-encoding and antitoxin components, respectively. Previous work showed that structures at the 5 end of RNA I are important in regulating its translation. The work presented here reveals that a stem-loop sequestering the Fst ribosome binding site is required for translational repression but a helix sequestering the 5 end of RNA I is not. Furthermore, disruption of the stem-loop prevented RNA II-mediated repression of Fst translation in vivo. Finally, although Fst-encoding wild-type RNA I is not toxic in Escherichia coli, mutations affecting stem-loop stability resulted in toxicity in this host, presumably due to increased translation.

Conjugation of the E. faecalis plasmid pCF10 is triggered in response to peptide sex pheromone cCF10 produced by potential recipients. Regulation of this response is complex and multi-layered and includes a small regulatory RNA, Anti-Q that participates in a termination/antitermination decision controlling transcription of the conjugation structural genes. In this study, the secondary structure of the Anti-Q transcript and its sites of interaction with its target, Qs, were determined. The primary site of interaction occurred at a centrally-located loop whose sequence showed high variability in analogous molecules on other pheromone responsive plasmids. This loop, designated the specificity loop, was demonstrated to be important but not sufficient for distinguishing between Qs molecules from pCF10 and another pheromone-responsive plasmid pAD1. A loop 5' from the specificity loop which carries a U-turn motif played no demonstrable role in Anti-Q-Qs interaction or regulation of the termination/antitermination decision. These results provide direct evidence for a critical role of Anti-Q/Qs interactions in posttranscriptional regulation of pCF10 transfer functions.

The par stability determinant is required for the stable inheritance of the plasmid pAD1 in its native host, Enterococcus faecalis. It is the only antisense RNA-regulated addiction module identified to date in grampositive bacteria. It encodes two small, convergently transcribed RNAs, RNA I and RNA II. RNA I encodes the Fst toxin and RNA II acts as the antitoxin by interacting with RNA I posttranscriptionally. As the toxinencoding component of the system, it is important that RNA I is more stable than RNA II. This study reveals that a helix sequestering the 5 end of RNA I plays a crucial role in maintaining the stability of the RNA I. An adjacent structure previously determined to regulate Fst translation was not required to enhance stability. Results indicated that endoribonuclease J2 contributes significantly to the degradation of a mutant disrupting the upstream helix (UH) of RNA I in Bacillus subtilis. Finally, it was shown that interaction with RNA II stabilized the UH mutant of RNA I.Addiction modules or postsegregational killing (PSK) systems stabilize plasmids within host cell populations by programming for death any daughter cell that loses the plasmid. PSKs are ubiquitous on low-copy-number plasmids and have been identified in both gram-negative and gram-positive bacteria (for reviews, see references 16 and 23). Addiction modules encode at least two components, a stable toxin and its unstable antidote, the antitoxin. In most cases, both toxin and antitoxin are proteins and toxin activity is regulated by direct interaction with its antitoxin. In a few cases, the antitoxin is a regulatory RNA that binds to the mRNA for the toxin and inhibits translation. Proper segregation of plasmid DNA ensures continued production of the labile antitoxin and suppression of toxin activity or translation of the toxin. Plasmid loss leads to degradation of the antitoxin and activation of toxin activity or translation, leading to cell death. Similar modules have been found on the chromosomes of most bacterial species, where they are believed to play a role in stress response (5, 9, 17).Two well-studied plasmid addiction modules have been shown to be regulated by an antisense RNA mechanism, the hok/sok system of Escherichia coli plasmid R1 and the par system of Enterococcus faecalis plasmid pAD1. Addiction modules present special problems for antisense RNA regulation, since the rapid degradation of the RNA-RNA complexes that occur in most such systems would leave no toxin message to be translated once the plasmid is lost. In the hok/sok system (14), this problem is solved by the accumulation of a pool of an inactive conformation of the hok mRNA that neither binds the sok antisense regulator nor allows ribosome binding (36). This pre-mRNA is then slowly degraded from the 3Ј end, triggering a conformational switch to a sok-and ribosome-binding form (11). If the plasmid is still present, sok binds rapidly via a U-turn motif located within one of the loops of the hok target (12) and the complex is rapidly degraded by RNase III (...