Abstract
Toxin–antitoxin (TA) systems are widespread in Gram-negative bacteria, and all systems identified to date encode a toxic protein and an unstable antitoxin, which may be in the form of either an antisense RNA (type I) or a second protein (type II). The enterococcal plasmid pAD1-encoded TA system (par), encoding the Fst toxin, was the first type I TA system identified in Gram-positive bacteria (Weaver et al., 1996). In a recent issue of Microbiology, Weaver et al. (2009) identified an additional eight pAD1-like TA systems. Individual fst-like genes were identified on the chromosomes of Enterococcus faecalis, Lactobacillus casei and Staphylococcus saprophyticus strains, plasmids from E. faecalis, Lactobacillus curvatus and Staphylococcus aureus, and a phage from Lactobacillus gasseri. It was also hypothesized that the small size of Fst-like toxins may cause the failure of other members of the family to be recognized and annotated. We have now addressed this issue through iterative TBLASTN searching of the translated NCBI nucleotide sequence database ().
Fst-like toxins were found to be prevalent in a diversity of Gram-positive bacteria. More than 200 additional toxins were detected in the database, and sequence similarity between previously identified Fst peptides and new family members in pairwise alignments was shown to be statistically significant using PRSS (E<0.02; Pearson, 1996). All are 27 to 35 residues in length and are predicted to contain a hydrophobic transmembrane domain. New Fst family members were found to be encoded chromosomally by Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Streptococcus pneumoniae, Streptococcus equi, Streptococcus suis, Streptococcus thermophilus, Streptococcus mutans and Listeria monocytogenes. Additional Fst-like toxins were also found on plasmids from Enterococcus faecalis, Enterococcus faecium, Staph. aureus, Staph. epidermidis, Staphylococcus warneri, Lactobacillus fermentum, Lactobacillus paracasei, Lactococcus lactis, Macrococcus caseolyticus and Carnobacterium divergens.
Analysis of the DNA sequence encompassing representatives of the newly identified Fst coding sequences from the various species revealed the presence of characteristic features shown to be important for function of the prototype pAD1 TA system (Greenfield & Weaver, 2000; Greenfield et al., 2000). Specifically, in addition to expression sequences for the toxin, they each possess sequences necessary for expression of an antisense RNA antitoxin, including a potential promoter closely resembling the σ70 consensus and a bidirectional terminator, converging towards the end of the toxin gene (Fig. 1). Thus, the resulting toxin mRNA and antitoxin possess a complementary thermodynamically stable stem–loop structure derived from the terminator; the loop residues have been shown to be important for interactions between the transcripts that result in the inhibition of pAD1 Fst toxin expression (Greenfield et al., 2001). Likewise, the directly repeated sequences DRa and DRb are readily identifiable. These tandemly arranged repeats overlap the toxin start codons, and are repeated downstream of the bidirectional terminators within each antitoxin gene, so as to provide a region of complementarity between the single-stranded 5'-tail of the antitoxin and the translation initiation region of Fst mRNA. Other intramolecular helices in the Fst mRNA have recently been shown to be important in the prototype pAD1 system: a 5' stem–loop structure that sequesters the Fst ribosome-binding site (comprising RBS and anti-RBS sequences), causing translational inhibition (Shokeen et al., 2008); and an upstream helix formed between complementary sequences at each end of the transcript (5' UH and 3' UH) that contributes to the stability of the toxin mRNA in vivo (Shokeen et al., 2009). The presence of sequences corresponding to all of these features in association with the newly identified toxin-coding sequences (Fig. 1) suggests a conserved mechanism of RNA–RNA interaction and control of toxin expression. It is therefore likely that most, if not all, of the Fst-like peptides detected are encoded by functional TA systems. Although the features above are well conserved, the nucleotide sequences of individual systems are quite divergent, even between multiple systems co-existing in the same strain, suggesting that cognate TA components might exhibit specificity.