A multistress responsive type I toxin-antitoxin system: bsrE/SR5 from the B. subtilis chromosome.

bsrE/SR5 is a type I TA system from prophage-like element P6 of the B. subtilis chromosome. The 256 nt bsrE RNA encodes a 30 aa toxin. The antitoxin SR5 is a 163 nt antisense RNA. Both genes overlap at their 3' ends. Overexpression of bsrE causes cell lysis on agar plates, which can be neutralized by sr5 overexpression, whereas deletion of the chromosomal sr5 copy has no effect. SR5 is short-lived with a half-life of ≈7 min, whereas bsrE RNA is stable with a half-life of >80 min. The sr5 promoter is 10-fold stronger than the bsrE promoter. SR5 interacts with the 3' UTR of bsrE RNA, thereby promoting its degradation by recruiting RNase III. RNase J1 is the main RNase responsible for SR5 and bsrE RNA degradation, and PnpA processes an SR5 precursor to the mature RNA. Hfq stabilizes SR5, but is not required for its inhibitory function. While bsrE RNA is affected by temperature shock and alkaline stress, the amount of SR5 is significantly influenced by various stresses, among them pH, anoxia and iron limitation. Only the latter one is dependent on sigB. Both RNAs are extremely unstable upon ethanol stress due to rapid degradation by RNase Y.

Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by RNases Y and J1.

Gene regulation accomplished by alternative folding of an mRNA is a widely used mechanism. Classical examples are the various transcriptional attenuation mechanisms that employ, for example, leader peptide translation, or binding of a modified protein, an uncharged tRNA or an antisense RNA to the 5' untranslated region of an mRNA. With the discovery of transcriptional and translational riboswitches, it became clear that small metabolites or even metal ions can also alter RNA secondary structures and, hence, gene expression. In addition, biophysical factors like temperature can affect RNA folding, as exemplified by RNA thermometers. We have investigated in detail the type I toxin-antitoxin system bsrG/SR4 from Bacillus subtilis. The antitoxin SR4 is a cis-encoded regulatory RNA that neutralizes BsrG toxin action. SR4 prevents toxin expression by promoting degradation of the toxin mRNA and inhibiting its translation. In addition, upon temperature shock the amount of toxin mRNA decreases significantly. Here, we demonstrate that heat shock induces a refolding in the central region of the toxin mRNA that makes it more accessible to degradation by RNases Y and J1. Furthermore, we show that BsrG might play a role at the onset of stationary phase, when the antitoxin SR4 can no longer prevent toxin synthesis.

In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis.

BsrE/SR5 is a new type I toxin/antitoxin system located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3' ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (kapp) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA system from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.

Against the mainstream: the membrane-associated type I toxin BsrG from Bacillus subtilis interferes with cell envelope biosynthesis without increasing membrane permeability.

Toxin-antitoxin loci, which encode a toxic protein alongside with either RNA or a protein able to counteract the toxicity, are widespread among archaea and bacteria. These loci are implicated in persistence, and as addiction modules to ensure stable inheritance of plasmids and phages. In type I toxin-antitoxin systems, a small RNA acts as an antitoxin, which prevents the synthesis of the toxin. Most type I toxins are small hydrophobic membrane proteins generally assumed to induce pores, or otherwise permeabilise the cytoplasmic membrane and, as a result, induce cell death by energy starvation. Here we show that this mode of action is not a conserved property of type I toxins. The analysis of the cellular toxicity caused by Bacillus subtilis prophage SPß-encoded toxin BsrG revealed that, surprisingly, it neither dissipates membrane potential nor affects cellular ATP-levels. In contrast, BsrG strongly interferes with the cell envelope biosynthesis, causes membrane invaginations together with delocalisation of the cell wall synthesis machinery and triggers autolysis. Furthermore, efficient inhibition of protein biosynthesis is observed. These findings question the simplistic assumption that small membrane targeting toxins generally act by permeabilising the membrane.

sRNAs in bacterial type I and type III toxin-antitoxin systems.

Toxin-antitoxin (TA) loci consist of two genes: a stable toxin whose overexpression kills the cell or causes growth stasis and an unstable antitoxin that neutralizes the toxin action. Currently, five TA systems are known. Here, we review type I and type III systems in which the antitoxins are regulatory RNAs. Type I antitoxins act by a base-pairing mechanism on toxin mRNAs. By contrast, type III antitoxins are RNA pseudoknots that bind their cognate toxins directly in an RNA-protein interaction. Whereas for a number of plasmid-encoded systems detailed information on structural requirements, kinetics of interaction with their targets and regulatory mechanisms employed by the antitoxin RNAs is available, the investigation of chromosomal systems is still in its infancy. Here, we summarize our current knowledge on that topic. Furthermore, we compare factors and conditions that induce antitoxins or toxins and different mechanisms of toxin action. Finally, we discuss biological roles for chromosome-encoded TA systems.

One antitoxin--two functions: SR4 controls toxin mRNA decay and translation.

Type I toxin-antitoxin systems encoded on bacterial chromosomes became the focus of research during the past years. However, little is known in terms of structural requirements, kinetics of interaction with their targets and regulatory mechanisms of the antitoxin RNAs. Here, we present a combined in vitro and in vivo analysis of the bsrG/SR4 type I toxin-antitoxin system from Bacillus subtilis. The secondary structures of SR4 and bsrG mRNA and of the SR4/bsrG RNA complex were determined, apparent binding rate constants calculated and functional segments required for complex formation narrowed down. The initial contact between SR4 and its target was shown to involve the SR4 terminator loop and loop 3 of bsrG mRNA. Additionally, a contribution of the stem of SR4 stem-loop 3 to target binding was found. On SR4/bsrG complex formation, a 4 bp double-stranded region sequestering the bsrG Shine Dalgarno (SD) sequence was extended to 8 bp. Experimental evidence was obtained that this extended region caused translation inhibition of bsrG mRNA. Therefore, we conclude that SR4 does not only promote degradation of the toxin mRNA but also additionally inhibit its translation. This is the first case of a dual-acting antitoxin RNA.

Type I toxin-antitoxin systems in Bacillus subtilis.

Type I toxin-antitoxin (TA) systems are widespread in bacteria and consist of a toxin-encoding mRNA and a partially overlapping antisense RNA that blocks expression of the toxin, either at the level of translation or by mRNA degradation. Four type I toxin families have so far been proposed in B. subtilis based on sequence similarity: TxpA/BsrG, BsrH/BsrE, YonT and YhzE and two (TxpA and BsrG) have been studied in some detail. Here we review what is known about these confirmed and putative toxin-antitoxin families in B. subtilis, their regulatory mechanisms, their potential roles and how they may link to the physiology of the cell.

BsrG/SR4 from Bacillus subtilis--the first temperature-dependent type I toxin-antitoxin system.

Here, we describe bsrG/SR4, a novel type I toxin-antitoxin system from the SPß prophage region of the Bacillus subtilis chromosome. The 294-nucleotide bsrG RNA encodes a 38-amino-acid toxin, whereas SR4 is a 180-nucleotide antisense RNA that acts as the antitoxin. Both genes overlap by 123 nucleotides. BsrG expression increases at the onset of stationary phase. The sr4 promoter is 6- to 10-fold stronger than the bsrG promoter. Deletion of sr4 stabilizes bsrG mRNA and causes cell lysis on agar plates, which is due to the BsrG peptide and not the bsrG mRNA. SR4 overexpression could compensate cell lysis caused by overexpression of bsrG. SR4 interacts with the 3' UTR of bsrG RNA, thereby promoting its degradation. RNase III cleaves the bsrG RNA/SR4 duplex at position 185 of bsrG RNA, but is not essential for the function of the toxin-antitoxin system. Endoribonuclease Y and 3'-5' exoribonuclease R participate in the degradation of both bsrG RNA and SR4, whereas PnpA processes three SR4 precursors to the mature RNA. A heat shock at 48°C results in faster degradation and, therefore, significantly decreased amounts of bsrG RNA.