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Strains carrying the Enterococcus (formerly Streptococcus) faecalis plasmid pADl responded to exogenous sex pheromone by inducing a number of gene products which facilitated mating. A 7-kilobase region of pADl was identified which contained genes that are important for the regulation of this response. Using the transposon Tn917-lac delivery vector pTV32Ts, we generated a number of fusions that allowed us to examine transcription in this region. At least three transcriptional units were identified by grouping fusions by their phenotype, direction of transcription, and response to pheromone. Transcription from one set of fusions was sensitive to the presence of pheromone. Analysis of the patterns of protein production previously shown to be induced in the presence of pheromone provided more information on the function of the genes of interest. We postulate the existence of two negative regulatory proteins that act coordinately to repress the pheromone response, one of which may be involved in sensing or transmitting the pheromone signal, and at least one positive regulatory protein whose synthesis is dependent on the presence of pheromone. In addition, the isolation of a relatively small deletion mutant capable of producing cAD1, the pheromone specific for pADl-containing cells, indicates that a factor(s) that is important for the shutdown of endogenous pheromone is also present in this region.

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Grampositive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Grampositive species. INTRODUCTIONToxin-antitoxin (TA) systems were first identified on bacterial plasmids, where they function as stability determinants by programming for death any daughter cells that fail to inherit a copy (for recent reviews see Gerdes & Wagner, 2007;Hayes, 2003). These systems, originally called post-segregation killing systems or addiction modules, encode a stable toxin and an unstable antitoxin. As long as the plasmid is properly inherited, antitoxin is continually replenished and the toxin remains inactive. If the plasmid is lost, the antitoxin is degraded and the toxin is free to exert its effect. Toxins have a variety of targets including gyrase (Bahassi et al., 1999;Jiang et al., 2002), ribosome-bound and free mRNA (Condon, 2006;Zhang et al., 2004), and the cell membrane (Gerdes et al., 1986). In most TA systems, both components are proteins, with the antitoxin targeted by a specific cellular protease (Gerdes et al., 2005). In two well-studied cases, the hok/sok system of Escherichia coli plasmid R1 (Gerdes et al., 1990) and the par system of Enterococcus faecalis plasmid pAD1 Greenfield et al., 2001, the antitoxin is a small regulatory RNA that represses the translation of the toxin. The RNA-regulated TA systems are sometimes referred to as type I TA loci while the proteinregulated systems are designated type II.Paradoxically, numerous TA systems have been identified on bacterial chromosomes and have been the subject of numerous recent reviews (Buts et al., 2005; EngelbergKulka et al., 2006;Fozo et al., 2008;Gerdes & Wagner, 2007;Gerdes et al., 2005;Van Melderen & Saavedra De Bast, 2009). Various roles have been either demonstrated or proposed for these systems, including stabilization of integrated mobile genetic elements, protection from plasmid-encoded addiction modules, programmed cell death, growth modulation during stress response, persistence, and developmental processes. It seems like...

Plasmid-free Enterococcus faecalis excrete peptides (sex pheromones) which specifically induce a mating response in strains harboring certain conjugative plasmids. The response is characterized by the synthesis of a "fuzzy" surface material, visible by electron microscopy, which is believed to facilitate the aggregation of donors and recipients. Transconjugants which receive a specific plasmid shut down the production of endogenous pheromone; however, they continue to produce pheromones specific for donors harboring different classes of plasmids. In this review, we summarize what is known about the biochemistry and genetics of this phenomenon. Some emphasis is given to the hemolysin plasmid pAD1 and the regulation of its conjugal transfer.

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.

The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multi-resistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

A 5-kbp region of pADi, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon.The complete DNA sequence of this =3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.

The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.