Identification And Funtionality Study Of Phd-Doc Toxin-Antitoxin System In Yersinia Pseudotuberculosis

Toxin-Antitoxin system(TAs)is widespread on the chromosomes and plasmids of bacteria and archaea.The system consists of two co-expressed genes,encoding stable toxin and unstable antitoxin,respectively.Toxins can usually affect important physiological processes of cells and ultimately lead to growth arrest or death.Antitoxins can protect bacteria by neutralizing the toxicity of toxins.The TA system has been considered as the gene module of the bacteria stress response,which plays an important role in cell adaptive,nutritional stress response,bacteria resistance,antibiotic resistance,and leading to persistence.Phd-Doc TA system including antitoxin Phd and toxin Doc,which belongs to Type? Toxin-Antitoxin system and was first discovered as a plasmid addiction module on Escherichia coli bacteriophage P1.Yersinia pseudotuberculosis(Yptb)is a zoonotic intestinal pathogen and serves as a model bacteria in microbiological research.At present,little is known about its stress tolerance and the related TA system in Yptb.In this study,we mainly identified the type ? Phd-Doc TA system and studied its function in Yptb stress tolerence.The detail of the results are as follows:1.Identification of Phd-Doc TA system in YptbBioinformatics analysis showed that there is a type ? Phd-Doc homology in Yptb highly conserved to bacteriophage P1,Escherichia coli,and bacteriophage P7.When the Doc toxin protein was expressed in Escherichia coli,the cell morphology was changed and cell growth was inhibited,which were rescued by the antitoxin protein Phd.Co-transcription experiments and bacteria two-hybrid experiments confirmed that the two genes phd and doc in Yptb are co-transcribed and the corresponding proteins showed strong interaction.In addition,EMSA and ?-galactosidase enzyme activity assay showed that the toxin Doc can enhance the binding activity of antitoxin Phd to their promoter,to inhibit the transcription activity of the promoter.The above results indicate that there is an active type ? Phd-Doc toxin-antitoxin pair in Yptb.2.The effect of Phd-Doc TA system on the growth of YptbWe constructed the mutant Yptb strains ?doc,?phd?doc and the corresponding complementary stains C?doc,C?phd?doc by homologous recombination and plasmid complementation,respectively.The growth curve determination showed that neither phd-doc nor doc,affects the growth of Yptb in nomal condition.In addition,the overexpression of Doc in Yptb and ?phd?doc showed on effect on the growth of Yptb.3.The effect of Phd-Doc TA system on Yptb stress responseIn this study,we explored the relationship between Phd-Doc TA system and stress response by growth curve and transcription level analysis.The results showed that under antibiotic and oxygen stresses,there was no difference in the growth of wild-type stains and the mutant strains ?doc,?phd?doc.But the transcription level of phd increased to varying degrees(1.5 to 2 times)compared with the control ones under above stress conditions.In addition,we found that the transcription level of phd was significantly increasing(25 to 30 times)under chloramphenicol stress,which was significantly higher than these under other stresses.This means that Phd-Doc TA system may be particularly sensitive to chloramphenicol stress.As a result,Phd-Doc does not affect Yptb's growth under different stress,however,it responds to the stresses at the transcription level,and particularly sensitive to chloramphenicol stress.4.Phd-Doc TA system affects the biofilm formation of YptbTA system was reported to be involved in biofilm formation.We furtherly explored the effect of the Phd-Doc TA system on the biofilm formation ability of Yptb under the condition of nutrient deficiency.The biofilm determination showed that the loss of Phd-Doc TA system can weaken the biofilm forming ability of Yptb under conditions of nutritional deficiency.In summary,we identified an active type ? Phd-Doc toxin-antitoxin pair in Yptb,and the physiological function of this TA system was preliminarily discussed,which enriched the understanding of Phd-Doc TA system.