Identification and characterization of a family of toxin–antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module

Abstract

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...

Characterization of the Effects of an rpoC Mutation That Confers Resistance to the Fst Peptide Toxin-Antitoxin System Toxin

“…Unlike another RNA-regulated peptide toxin family, the Hok/Gef toxins, overexpression of Fst/Ldr toxins does not result in the formation of "ghost cells," in which the cell center becomes translucent and the cell material appears to condense at the poles (21,22). Rather, the primary effect of overexpression of Ldr in Escherichia coli (23) and Fst in E. faecalis, Bacillus subtilis, Staphylococcus aureus, and E. coli (11,24) is the condensation of the nucleoid. In E. faecalis, nucleoid condensation is accompanied by aberrant chromosome segregation, irregular division septum placement, and loss of membrane integrity (24).…”

“…These peptides are characterized by the presence of a hydrophobic putative transmembrane domain and a highly charged C-terminal tail. Mutagenic analysis of Fst indicated that the hydrophobic domain is essential for toxicity while the C-terminal domain is dispensable (11). Recent nuclear magnetic resonance structural analysis of Fst in a membrane mimetic confirmed that amino acids 3 to 26 form a transmembrane domain with the relatively unstructured C-terminal tail protruding, likely into the cytoplasm (20).…”

“…Targets of PSK systems include DNA gyrase (5, 6), ribosome-bound or free mRNA (7), peptidoglycan synthesis (8), and the cell membrane (9). Numerous homologs of type I and type II PSK systems have been identified on the chromosomes of many bacterial species (10,11,12). The roles of these chromosomally encoded systems are diverse and, in some cases, poorly defined or controversial (9,10,13,14,15).…”

“…When translated, RNAI produces a 33-amino-acid peptide toxin called Fst (18). Fst is the founding member of the Fst/Ldr peptides, a large family of RNA-regulated toxins that are widespread on plasmids and chromosomes of low-GϩC Gram-positive bacteria as well as the Gram-negative enterobacteria (11,12,19). These peptides are characterized by the presence of a hydrophobic putative transmembrane domain and a highly charged C-terminal tail.…”

Persistent virus and addiction modules: an engine of symbiosis

“…Indeed, the E. coli PAR addiction module which expresses intragenic stem-loop RNAs that can regulate translation might be the best model for understanding how non-coding RNA can regulate addiction [31]. Many related loci that also use RNA are known [33]. Also, noncoding antisense RNA is used as a component of widely distributed abortive infection systems (Abi) of bacteria that counteracts a wide array of virus [34].…”