Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein–protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

Chromosomally-encoded Type II Toxin-Antitoxin operons are ubiquitous in bacteria and archaea. Antitoxins neutralize the toxic effect of cognate Toxins by protein-protein interactions and sequestering the active residues of the Toxin. Toxins target essential bacterial processes, mostly translation and replication. However, one class apart is constituted by the PezAT pair because the PezT toxin target cell wall biosynthesis. Here we have examined the role of the pezAT toxin-antitoxin genes in its natural host, the pathogenic bacterium Streptococcus pneumoniae. The pezAT operon on Pneumococcal Pathogenicity Island 1 was deleted from strain R6 and its phenotypic traits were compared with those of the wild type. The mutant cells formed shorter chains during exponential phase, leading to increased colony-forming units. At stationary phase, the mutant was more resilient to lysis. Importantly, the mutant exhibited higher resistance to antibiotics targeting cell walls (β-lactams), but not to antibiotics acting at other levels. In addition, the mutants also showed enhanced genetic competence. We suggest that PezAT participates in a subtle equilibrium between loss of functions (resistance to β-lactams and genetic competence) and gain of other traits (virulence).

Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between four to ten putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary19A-6 which contains all possible ten TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.

Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the −10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.