Functional Identification Of The Transcription Factor CysB Interacting With The TmRNA Promoter Of Aeromonas Veronii C4

Aeromonas veronii(A.veronii)is a co-pathogenic bacterium of humans and fish that infects economic fish and causes diseases such as septicemia,causing huge losses to the aquaculture industry.There is a trans-translation system mediated by transfer-messenger RNA(tmRNA)and small protein B(SmpB),which makes the bacteria resistant to environmental stresses by ribosome rescue.In the signal transduction pathway,previous studies were limited to the downstream of tmRNA regulation,and there was no mining and reporting of the upstream signal of tmRNA was regulated.In this study,A.veronii C4 was selected as the target and tmRNA as entry point for researching on the upstream transcription factors of tmRNA.The results are as follows:First,LysR type transcription factor CysB was predicted to regulate tmRNA promoter(PssrA)by convolutional neural network.Secondly,the interaction between PssrA and CysB was verified.The transformants coexpressing CysB and PssrA were able to grow on resistance screening plates with addition of 10 mM 3-AT and streptomycin,indicating the interaction between CysB and PssrA.In electrophoretic shift assay(EMSA)experiment,CysB-PssrA complex formed after adding 30 ng CysB protein.While once adding excessive specific competition DNA,the hysteresis band disappeared,indicating that CysB-PssrA interaction is specific.Thirdly,how CysB regulates tmRNA was explored.In the fluorescence intensity measurement assay,when PssrA was co-expressed with CysB,the fluorescence value of the strain was significantly increased,which suggested that CysB positively regulated tmRNA to increase eGFP expression.In real-time fluorescent quantitative PCR(RT-qPCR)experiment,the relative expression of tmRNA was significantly reduced after cysB was knocked out.Collectively,these results suggested that CysB positively regulates tmRNA.Then,key sites of CysB binds to PssrA were explored.PRODORIC2 is a prokaryotic regulatory database,which finds the general formula of CysB binds DNA sequence5’-NDDDNGWTATNNNNNNNNNHWWNBNMNNHWK-3’.Fourthly,compared with PssrA sequence,truncated PssrA retained the region where CysB binds to PssrA.The result of EMSA showed that PssrA still binded to CysB after truncation.In order to find out the key sites of interaction between CysB and PssrA,the point mutation was carried out at the sequence of PssrA.These results of bacterial one-hybrid and fluorescence intensity determination assay showed that ATTTTT and ACAAT were the key sites of CysB binds to PssrA.Finally,the effects of tmRNA regulated by CysB on the Swimming and biofilm were investigated.The transcriptome analysis showed that genes related to flagella synthesis were down-regulated.The swimming experiment showed that swimming ability was decreased after ssrA or cysB deleted.Biofilm experiments showed that the biofilm formation ability was also decreased after ssrA or cysB deleted.All these results suggested that the regulation of tmRNA by CysB affected the bacterial swimming and biofilm.In this study,the upstream transcription factors regulating tmRNA in Aeromonas veronii were investigated for the first time.The regulation of tmRNA by CysB decreased the swimming and the formation of biofilm.It provides a new idea for aquaculture to treat with the related diseases caused by A.veronii.