SummaryHere, we describe bsrG/SR4, a novel type I toxinantitoxin system from the SPb 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 3Ј 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 3Ј-5Ј 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.

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.

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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.