BsrE/SR5: ein neues TypI-Toxin/Antitoxin-System in Bacillus subtilis: Toxinlokalisierung, Identifizierung der zellulären Toxin-Targets, Aufklärung des Wirkungsmechanismus des Antitoxins, biologische Funktion des Systems
Zusammenfassung der Projektergebnisse
In the frame of the present project, we have investigated the type I toxin-antitoxin system bsrE/SR5 both in vitro and in vivo. The secondary structures of both interacting RNAs and of their complex were experimentally determined, the interaction pathway of both RNAs elucidated and minimal regions for effective interaction narrowed down. These in vitro data were corroborated by in vivo data. The mechanism of action of the RNA antitoxin SR5 was elucidated: SR5 exerts its inhibitory function by promoting bsrE mRNA degradation by the double-strandspecific RNase III, but does not impact bsrE translation. This is in contrast to the type I TA system bsrG/SR4 which was investigated in our group before. bsrE/SR5 is a multistress-responsive TA system, but only under anaerobic conditions, an excess of the toxin-mRNA over the amount of the antitoxin SR5 was measured. Therefore, we conclude that expression of the bsrE mRNA, and, consequently, the toxin BsrE is only possible under oxygen limitation. We hypothesize that under anaerobic conditions, bsrE might be expressed in single cells leading to cell death or growth retardation to allows the remaining living cells to adapt to this environmental change. The in vivo study of a third type I TA system from B. subtilis, yonT/yoyJ/SR6 revealed that the antitoxin SR6 regulates two toxins: By complementary base-pairing between the 3’ ends of SR6 and yonT mRNA, SR6 promotes degradation of yonT mRNA by RNase III. In addition, by base-pairing between the 5’ ends of SR6 and yoyJ mRNA, SR6 most probably inhibits translation of yoyJ mRNA encoding the much weaker, but also very weakly expressed second toxin YoyJ. Like bsrE/SR5, yonT/yoyJ/SR6 also responds to various stresses. Only under heat-shock conditions, the amount of yonT mRNA is higher than that of its neutralizing antitoxin SR6, suggesting that this TA system might play a role under heat-shock or generally high temperatures. To complete our previous studies of the type I TA system bsrG/SR4 we unraveled the molecular background of the 4-fold decreased stability of bsrG mRNA under heat-shock: Heat-shock (37° to 48° or 55 °C) causes melting of the highly compact secondary structure of bsrG mRNA in the central region which allows the main endoribonuclease RNase Y and the endo-/5’-3’ exo-RNase J1 to cleave the toxin mRNA. Furthermore, we determined the SR4/bsrG mRNA ratio over growth and found that at transition from logarithmic to stationary growth, the amount of the bsrG toxin mRNA exceeds that of the neutralizing antitoxin SR4 which allows expression of the toxin. Therefore, we conclude that bsrG/SR4 might play a role in stationary phase adaptation of B. subtilis. Due to the impossibility to perform the planned collaborations in the frame of type I TA systems, we started a new project: We investigated the RNA chaperone CsrA, for which so far only one target, hag mRNA was known in B. subtilis, and no interacting sRNA had been discovered. Our results reveal a completely new function for CsrA not only in B. subtilis, but in bacteria in general: CsrA can promote the interaction between a small regulatory RNA, SR1, and its primary base-pairing target, ahrC mRNA. This was a function until now only known for Hfq and ProQ from Gram-negative bacteria. In the frame of a new DFG-project, we will investigate if CsrA plays a more global role in Bacillus subtilis.
Projektbezogene Publikationen (Auswahl)
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A multistress responsive type I toxin-antitoxin system: bsrE/SR5 from the B. subtilis chromosome. RNA Biol. 13: 511-523 (2016)
Müller, P., Jahn, N., Ring, C., Maiwald, C., Neubert, R., Meißner, C. and Brantl, S.
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Heat-shock-induced refolding entails rapid degradation of bsrG toxin mRNA by RNases Y and J1. Microbiology 162: 590-599 (2016)
Jahn, N. and Brantl, S.
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In vitro characterization of the type I toxin-antitoxin system bsrE/SR5 from Bacillus subtilis. J. Biol. Chem. 291: 560-571. (2016)
Meißner, C., Jahn, N. and Brantl, S.
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Bacillus subtilis type I antitoxin SR6 promotes degradation of toxin yonT mRNA and is required to prevent toxic yoyJ overexpression. Toxins (Basel) 10: 74 (2018)
Reif, C., Löser, C., and Brantl, S.
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A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis. RNA Biol. 16: 972-987 (2019)
Müller, P., Gimpel, M., Wildenhain, T., and Brantl, S.
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Toxin-antitoxin systems in Bacillus subtilis. Toxins (Basel) 11: 262 (2019)
Müller, P., and Brantl, S.
