Identification and characterization of mazEF family type II toxin-antitoxin systems of Leuconostoc pseudomesenteroides L64

Abstract:

Objective To identify mazEF family type II toxin-antitoxin systems of Leuconostoc pseudomesenteroides L64 and to elucidate the molecular roles of the mazEF systems in the host exposed to environmental acid stress.Methods Putative MazF toxins were induced alone or co-expressed with their cognate antitoxins in Escherichia coli. The toxic effect of MazF on bacterial growth and the antitoxic effects of cognate antitoxins were examined. The lacZ reporter system and electrophoretic mobility shift assay (EMSA) were used to decipher the auto-regulation mechanism of the mazEF system in vivo and in vitro. The putative target genes regulated by MazE were predicted and validated through in vivo and in vitro experiments.Results Among the three putative mazEF systems in L. pseudomesenteroides L64, mazEF1-Leup (OYT_01690-OYT_01685) encoded a functional type II toxin-antitoxin system. MazE1-Leup (OYT_01685) inhibited mazEF1-Leup transcription by binding to the palindromic sequence (TAACAaaatgTGTTA) in the promoter. In addition, MazE1-Leup inhibited transcription of the dlt-acpS-alr operon by binding to the similar palindromic sequence (TAACAtattgaaatatatgTGTTA) in the promoter of dlt-acpS-alr.Conclusion mazEF1-Leup (OYT_01690-OYT_01685) encodes a functional mazEF family type II toxin-antitoxin system. Beyond regulating its own operon, MazE1-Leup regulates the transcription of dlt-acpS-alr and finally assists L. pseudomesenteroides L64 in response to low acid stress.