Research On Identification And Function Of A New Type Of Holin-endolysin Lysis System In Acidovorax Orvzae Phage AP1

Bacterial brown stripe caused by Acidovorax oryzae is an important Bacterial disease and has caused serious economic losses to rice production.As natural enemies of bacteria,phages encode different lysis systems,which play an important role in killing bacteria efficiently and specifically.Therefore,the in-depth study on phage lysis system of rice bacterial brown stripe disease not only provides a new vision for fully revealing its lysis mechanism,but also provides a new way for the prevention and control of rice bacterial brown stripe disease by using phage lysis system.First,based on the previous isolation and determination of the whole genome sequence of Acidovorax oryzae phage API,we identified two adjacent and cleavage-related genes through bioinformatics analysis:holin?HolAP?and endolysin?LysAP?.HolAP is found to be upstream of LysAP.And it is the member of the phage holin superfamily Phage_holin₂₃ by conservative domain analysis.;Prediction of the protein transmembrane shows that HolAP has a transmembrane region,and the N-terminus is distributed outside the cell membrane,while the C-terminus in the cytoplasm,which indicates the structural features of type ? holin.Moreover,LysAP is predicted to belong to Lysozyme_like_superfamily of the bacteriophage endolysin family based on conservative domain analysis;In addition,endolysin is predicted to has a transmembrane region at the C-terminus according to protein transmembrane analysis,indicating that there may exist a membrane localization;and the signal peptide predicts that the protein does not contain a signal peptide,indicating that it can not directly secrete into periplasm of the cell to cause lysis.Secondly,this study determined the function of holin HolAP.Overexpression of HolAP in Escherichia coli was determined and the bacteria growth results indicate that the OD600 value of bacteria did not decrease and the growth of bacteria was not affected;The bacteria were judged to be living by showing green fluorescence;Observation of TEM revealed that the cell wall and cell membrane of the bacteria were slightly shrivelled,the intracellular substance density decreased,and the bacteria body color became lighter,indicating that HolAP expression could cause intracellular material leakage;Adding O-nitrophenol-galactosidase?ONPG?to determine the ?-galactosidase in the bacterial supernatant,the color of the solution does not change,indicating that the ?-galactosidase is not secreted into the extracellular environment through the cell membrane;For the detection of membrane protein and intracellular protein by western blotting,only HolAP protein bands were detected in the membrane components,while no bands in the intracellular components,indicating that HolAP was located on the cell membrane of bacteria.Thus,HolAP was preliminarily confirmed to have the characteristics of holin.Thirdly,this study determined the function of LysAP.Overexpression of LysAP in E.coli was determined and the bacteria growth results indicated that the OD600 value of bacteria decreased;the bacterial solution gradually became clear;and bacterial fragments appeared at the bottom of the test tube,indicating that the bacteria began to lysis.Under the fluorescence microscope,the bacteria showed red fluorescence,which was determined as dead bacteria,indicating that the expression of LysAP could kill bacteria.Tested the?-galactosidase activity of the bacterial supernatant,after adding ONPG,the solution changes from colorless to yellow,showing that the intracellular(3-galactosidase can leak into the extracellular supernatant through the cell membrane;Observation of TEM showed that the cell membrane of the bacteria was severely wrinkled;the cell wall structure became irregular;and the contents of the cell were leaking,which further demonstrated the lysis ability of LysAP.Based on the previous bioinformatics analysis,it was predicted that LysAP had no signal peptide,but there was a transmembrane region at the c-terminus.By constructing the C-terminus deletion mutant and measuring the influence of its overexpression on bacterial growth,it was found that after the deletion of the transmembrane region,the OD600 value of bacteria did not decrease and the growth was not affected;SDS-PAGE found that LysAP could not be anchored to the cell membrane due to the absence of C-terminal transmembrane region,which was largely concentrated in the cytoplasm,so that it could be purified.Therefore,LysAP was confirmed to encode endolysin,and its c-terminal transmembrane region was critical for protein localization and function.Finally,HolAP and LysAP had synergistic effects on bacteria lysis.The bacterial colony turned blue according to the two-hybrid experiment,which showed the interaction between HolAP and LysAP.Through the construction of co-expression plasmids of HolAP and LysAP,the growth curve of bacteria indicated that the OD600 value decreased rapidly;the bacterial fluid became clear and sticky;and a large number of bacterial fragments appeared at the bottom of the test tube.Under the fluorescence microscope,the bacteria showed red fluorescence,which was determined as dead bacteria;Gram staining showed that the baculosity of the bacteria changed from short to round,and the cell boundary was blurred.Transmission TEM showed that the bacterial cell membrane was severely wrinkled,the cell wall structure became irregular,the contents of the cell leaked,and even caused cavitation.ONPG was added to determine the ?-galactosidase in the bacterial supernatant,and the solution changed from colorless to dark yellow,which darker than the supernatant expressing LysAP alone,thus confirming the synergistic effect of HolAP and LysAP of bacteria lysis.