RNA structures regulating stable inheritance or conjugation of enterococcal plasmids, pAD1 and pCF10.

Enterococcus faecalis is a natural inhabitant of the mammalian gastrointestinal tract. It is an opportunistic pathogen that is a major cause of urinary tract infections, bacteremia, and infective endocarditis. As a nosocomial pathogen it has become refractory to most therapeutic options. Plasmids are important for both dissemination of antibiotic resistance and virulence factors in this organism. The par stability determinant of the E. faecalis plasmid pAD1 is the only antisense RNA-regulated addiction module identified in Gram-positive bacteria (Weaver and Trittle, 1994). Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, that function as the toxin (Fst)-encoding and antitoxin components of the system, respectively. A helix sequestering the 5' end of RNA I is critical for stability while and a second structure, a stem loop, is involved in translational repression. Fst affects cell division and chromosome partition in Gram-positive hosts (Patel and Weaver, 2006), but Gram-negative hosts were believed to be resistant. However, a mutation that disrupts the RNA stem-loop regulating fst translation led to nucleoid segregation defects in Escherichia coli similar to those observed in Gram-positive hosts. pCF10 is a pheromone inducible enterococcal conjugative plasmid. Previous research has elucidated the highly specific conjugative initiation process wherein the import of pheromone is enhanced by a protein, PrgZ (Bae et al., 2004). The pheromone then binds to a molecular switch, PrgX (cytoplasmic receptor protein), and triggers the complex cascade of transcriptional and post-transcriptional events, which leads to expression of the prgQ operon. The prgQ operon encodes conjugative transfer of proteins and regulatory factors. Qa is a negative regulator, which interacts with nascent prgQ transcript (Qs) and terminates its transcription before the expression of downstream conjugative loci. The Structural and mutational analyses revealed that 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 pAD1. A loop 5' to the specificity loop which carries a U-turn motif played no demonstrable role in Qa-Qs interaction. These results provide direct evidence for a critical role of Qa/Qs interactions in posttranscriptional regulation of pCF10 encoded conjugative machinery.