Characterization And Functional Investigation Of Type ? Toxin-antitoxin System DinJ-YafQ In Tetragenococcus Halophilus

Tetragenococcus halophilus is a moderately halophilic lactic acid bacterium widely used in high-salt food fermentation because of its coping ability under salt stress and good fermentation characteristics.In the process of industrial fermentation,T.halophilus is exposed to complex environmental conditions,such as high salt concentration,low p H,nutrient stress,high temperature,oxidative stress,which will affect physiological metabolism and survival of microorganisms.Bacterial toxin-antitoxin(TA)systems are widely distributed and confirmed to regulate the physiological behavior of cells by regulating gene expression under stress responses.Similar to other bacteria,T.halophilus genome contains multiple toxin-antitoxin systems,but their characteristics and physiological functions have never been explored.By bioinformatics analysis of T.halophilus CICC 10469 genome,16 putative type II TA systems were predicted and we selected TEH?RS02450-TEH?RS02455 and TEH?RS06855-TEH?RS06860 for further study to explore the effect of type II toxin-antitoxin systems on the physiological and biochemical processes of T.halophilus.TEH?RS02450 and TEH?RS02455 are co-transcribed and encode the bona fide DinJ-YafQ system,named Din J₁-Yaf Q₁^tha.Overexpression of Yaf Q₁^thacaused severe growth inhibition of Escherichia coli,while the toxic effects of Yaf Q₁^thacould be eliminated by introducing of Din J₁^thaand forming of Din J₁-Yaf Q₁^thacomplex.Based on gfp reporter gene systems and electrophoretic mobility shift assays,we showed that Din J₁^thaand Din J₁-Yaf Q₁^thacomplex could bind to the din J₁-yaf Q₁^thapromoter region to autoregulate their transcription,the latter showed more effective inhibitory effect of din J₁-yaf Q₁^thapromoter.The toxin protein Yaf Q₁^thaacted as a ribonuclease,and its enzymatic activity was blocked through formation of the Din J₁-Yaf Q₁^thacomplex,indicating that Yaf Q₁^thacan function as RNase decomposing gene expression profile when T.halophila copes with stress conditions.Results also demonstrated that din J₁-yaf Q₁^thainvolved in plasmid maintenance and stress response,and its transcriptional level was regulated by various stresses,especially under osmotic stress and acidic stress,which was significantly upregulated?It was predicted that TEH?RS06855-TEH?RS06860 encodes the second pair of DinJ-YafQ system.After further gene structure analysis,there is only a single antitoxin Din J,named Din J₂^tha,due to the loss of toxin Yaf Q.Interestingly,when Din J₂^thaand Yaf Q₁^thawere co-expressed,growth arrestment caused by Yaf Q₁^thawas abolished and protein-protein binding was found,suggesting that antitoxin Din J₂^thapresented cross-interaction with noncognate toxin Yaf Q₁^tha.Further analysis also found cross-regulation,Din J₂^thawas able to bind directly to the promoter sequence of din J₁-yaf Q₁^thaoperon and repressed its expression.We found that the amino acid similarity between Din J₁^thaand Din J₂^thais low,phylogenetic analysis showed that those proteins are in distantly separate clusters.We propose that din J₁^thaand din J₂^thahave been acquired from different evolutionary origins and inserted into genome through horizontal gene transfer.Collectively,the characterization of DinJ-YafQ^thamay be helpful to investigate the characteristics and key roles of TA systems in T.halophilus and serves as a foundation for further researches.