Lysis Mechanism Of Phage Endolysin And Its Antioxidant Function In Host Acidovorax Oryzae

Endolysin is a peptidoglycan hydrolase encoded by bacteriophages that helps itself release offspring by destroying host cells.It plays an important role in the long-term interaction and evolution of bacteriophage and bacteria.It has been reported that host bacteria can integrate the prophage genes by horizontal gene transfer and lysogenic transformation to obtain new phenotypes except immunity,and can reproduce normally without cleavage.Our previous studies found that the bacteriophage AP1 uses LysAP to lyse Acidovorax oryzae RS-2.However,few reports indicated that host bacteria also encode LysAP-like protein and revealed its biological function.To the best of our knowledge,this is the first report to discover a protein(LysAo)which is highly homologous to endolysin(LysAP)of bacteriophage AP1 in host Acidovorax oryzae RS-2.Moreover,the coexistence of endolysin in bacteriophage and host bacteria also highlights the importance of endolysin in bacteriophage-bacteria interactions,which make it urgent to understand the function of bacteriophage endolysin-like protein in host bacteria.In order to identify the mechanism and its biological function in host,laying a foundation for the understanding of the interaction between bacteriophage and rice pathogenic bacteria,following work was carried out in this study based on the promising results of our preliminary research:To investigate the transport pattern of endolysin LysAP in bacteriophage and the amino acid sites affecting its lytic efficiency,firstly the bacterial growth curve was measured after the addition of sodium azide and we observed that the bacterial lysis was inhibiting.It was preliminarily confirmed that lys AP was transported to the inner membrane by Sec protein.Moreover,we mutated LysAP at the amino acid site and conducted the research.The result showed that the mutant at conservative site(LysE15A)had a significant inhibitory on bacterial lysis,whereas the mutant of one cationic amino acid near transmembrane domain(LysR222A)showed weak inhibitory.Finally,western blot showed there was no protein band in cytoplasm of LysR222 A sample,indicating that the charge change at R222 had no effect on membrane anchoring of LysAP.To investigate the biological function of LysAo,first of all,bioinformatics prediction analysis showed that LysAo belong to Lyz＿like＿superfamily and had highly similarity in protein structure with LysAP.Then,we constructed overexpression plasmid p ET-28a-lysAo and translated it to BL21(DE3).By measuring the growth curve,conducting fluorescent staining test,gram staining test,transmission electron microscopic observation andβ-galactosidases activity test,the results showed that the overexpression of LysAo will lead to the disintegration of bacterial cell wall and the inhibition of the growth rate,which make sure that LysAo protein has activity of lysing bacteria.Moreover,the mutant strain with lysAo gene deletion was constructed and compared with wild type strain in bacterial growth,swimming,biofilm-forming,secretion of exopolysaccharide,extracellular enzyme activity,resistance to bacteriophage infection,hydrogen peroxide tolerance.Results showed that tolerance of mutant was significantly weaker than that of wild type.Lastly,q PCR test showed that hydrogen peroxide could stimulate the expression of lysAo encoded gene.Detecting bacterial hydrogen peroxide scavenging ability,the wild type was found to have a stronger ability to remove hydrogen peroxide than that of the mutant while the three major antioxidant enzymes had no significant difference.And pathogenicity test of rice showed that the gene deletion of lysAo reduced the pathogenicity of the bacteria RS-2.Overall,on one hand,this study revealed the lytic mechanism of LysAP which laid a theoretical foundation for the application of LysAP.On the other hand,LysAo,a bacteriophage endolysin-like protein with high homology to LysAP,was found in the host bacteria.It is proved that LysAo is related to the resistance of bacteria to oxidative stress and pathogenicity to rice plants,which will not only greatly enrich the pathogenic mechanism of rice pathogenic bacteria,but also provide a new insight for us to understand the interaction of bacteriophage-bacteria.