Identification And Functional Study Of HipBA Toxin-Antitoxin System In Acidovorax Citrulli

Bacterial fruit blotch(BFB)is a quarantined,destructive,seed-borne bacterial disease caused by gram-negative bacerium Acidovorax citrulli,and has caused serious damages and great economic loss to the watermelon industry in China.The Toxin-Antitoxin(TA)system is widely present on the chromosomes of bacteria.The TA system is composed of two genes,toxin and antitoxin,encoding stable toxin protein and unstable antitoxin protein,respectively.They usually interact to each other to protect cells from toxins.TA system,as a bacterial stress response system,plays an important role in harsh environments,antibiotic tolerance and other situations.TA systems are classified into eight types(I-VIII)based on their antitoxin properties and how they neutralize with homologous toxins.The HipBA belongs to the Type II TA system.Due to that TA system may play a role in the virulence and dormancy of human pathogens,the research on TA system in human pathogens has received a lot of attentions.However,the research on TA system in plant pathogens is still lacking,and there are few reports on the bacterial plant pathogen A.citrulli.In this study,we identified the toxin-antitoxin system encoded by hipBA in Aac5 of the A.citrulli,and discussed whether HipBA as a TA system,was involved in the stress tolerance of Aac5.Specific research results are as follows:1.Identification of HipBA toxin-antitoxin system in A.citrulliBioinformatics analysis has identified a putative TA system in the genome of A.citrulli AAC00-1,which is homologous to the identified HipBA TA system in other bacterial species.The predictable protein HipA was heterologous expressed in Escherichia coli,and the growth rate was also determined.The results showed that the HipA had signigicant inhibitory effect on the growth of E.coli.At the transcription level,the open reading frames of hipA and hipB were located on the same m RNA,indicating that the two genes were co-transcripted;The two proteins encoded by hipA and hipB can interact directly in vivo through bacterial two-hybrid experiment;Electrophoretic Mobility Shift(EMSA)Assay experiment showed that the antitoxin HipB can bind specifically to its own promoter.These results indicate that Aac5 strain of A.citrulli has an active type II HipBA TA system.2.Transcriptional analysis of hipA and hipB in A.citrulli under different environmental stressIn this study,changes in transcription levels of hipB and hipA of Aac5 strain were detected under different stress conditions.To discuss whether the HipBA TA system responds to stress,the growth ability and transcription level of target genes under different p H and antibiotic(chloramphenicol)pressure were measured.The results showed that the expressions of hipA and hipB were up-and down-regulated after treated with stress conditions,indicating that HipBA TA system responded to chloramphenicol stress and p H stress at the transcriptional level.The transcription levels of hipA and hipB in the Aac5 were measured at different time points after inoculation.The results showed that hipA and hipB were activated during the host-pathogen interaction,indicating that HipBA TA system was involved in the pathogen-host inbteractions.In conclusion,this study identified an active type II toxin-antitoxin system,HipBA,on the genome of Aac5,and the biological function of TA system was preliminarily explored,which enriched the understanding of TA system in plant pathogens.