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 0 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 halflife 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 0 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.

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 (k app ) 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.Small regulatory RNAs (sRNAs) 3 are key players in bacterial post-transcriptional gene regulation and have been discovered in a plethora of species (reviewed in Refs. 1-3). They employ either RNA/RNA base pairing or protein binding to inhibit or activate target gene expression. A special case of base pairing sRNAs is type I antitoxins that interact with complementary mRNAs encoding small toxic peptides (reviewed in Refs. 4 and 5).Originally, type I toxin-antitoxin (TA) systems were discovered on plasmids (e.g. hok/Sok on Escherichia coli plasmid R1 (6, 7) or fst/RNAII on Enterococcus faecalis plasmid pAD1 (reviewed in Refs. 8 and 9)), in which they act as postsegregational killing systems. Subsequently, many chromosome-encoded type I TA systems were found and investigated, e. (14). They are arranged as overlapping, convergently transcribed gene pairs or as divergently transcribed gene pairs located apart. The interaction between RNA antitoxin and toxin mRNA either inhibits translation or facilitates degradation of the toxin mRNA (5). One exception is bsrG/SR4, whose antitoxin SR4 is bifunctional: it promotes degradation of the toxin mRNA and inhibits toxin translation by inducing a structural alteration around the bsrG ribosome binding site (RBS) (15). Another exception is fst/RNAII from E. faecalis, in which antitoxin binding yields a complex that stabilizes both RNAs but prevents toxin translation (16).Some chromosome-encoded type I TA systems are involved in persister formation (17-20), whereas others are involved in recycling of damaged RNA (21), DNA recombination (22), or antibiotic resistance (23). Of 14 predicted B. subtilis type I systems, 5 are located on prophages and might be required for their maintenance or overcoming metabolic or environmental stress (24). Recently, we published the first temperature-dependent type I TA system bsrG/SR4 and investigated it both in vivo and in vitro (14, 15). The 38-amino acid hydrophobic toxin BsrG causes mem...