Effect Of Quorum Sensing (QS) On Endogenous Colonization Of Burkholderia Pyrrocinia JK-SH007

Quorum sensing(QS)exists widely in Gram negative bacteria(G^-)and Gram positive bacteria(G⁺).It is an important way for exchanging information among bacteria,between species cells and between bacteria and hosts,and regulates many important physiological functions of bacteria.Burkholderia pyrrocinia JK-SH007 is an endophytic control bacteria that can effectively control poplar ulcer disease,and clarifying its QS system and its endogenous colonization mechanism will help reveal its biocontrol mechanism.The main findings are as follows:1.The crude extracts of AHLs and DSF of strain JK-SH007 were obtained by ethyl acetate extraction and rotary evaporation,and QS indicator bacteria and liquid chromatography were used to determine that strain JK-SH007 has two QS systems,respectively.For the Cep IR system and DSF system,the types of QS signal substances generated are C₈-HSL and BDSF,respectively.2.The effects of QS signalling substances C₈-HSL and BDSF on the biofilm formation of strain JK-SH007 and its colonization ability in poplar seedlings were studied using crystal violet staining,bacterial cell recovery,and GFP labeling techniques.The results showed that C₈-HSL could promote the formation of the biofilm and the endogenous colonization ability of the strain JK-SH007 in a certain concentration range.C₈-HSL is closely related to the endogenous colonization ability of the strain JK-SH007 in poplar trees.C₈-HSL can enhance the endogenous colonization effect of strain JK-SH007.3.Through cloning and analysis of the QS gene of JK-SH007,homologous sequences of QS signal molecules encoding genes Cep I and Rpf F existed in JK-SH007.Use bioinformatics software to analyze the function and characteristics of genes.The open reading frame of the Cep I gene is 609 bp,encoding 202 amino acids,the p I is 5.82,and it contains 9phosphorylation sites.Rpf F protein has a molecular weight of 33980.85 Da,a theoretical isoelectric point of 5.94,and contains 10 phosphorylation sites.Both Cep I and Rpf F proteins have no signal peptide and belong to hydrophilic proteins.The secondary structure shows that the maximum structural elements of Cep I and Rpf F proteins include alpha-helix(Beta bend),beta bend,and extended chain(Extended strand)and random coils.Among Cep I proteins,the ratio of random coils is the highest,reaching 42.08%of the total secondary structure;the ratio of?-turn angle is the smallest,accounting for 5.94%of the total secondary structure;?-helix accounts for 34.65%of the total secondary structure;?-sheet accounts for the total Secondary structure 17.33%.The secondary structure prediction results of the Rpf F protein showed that the?-helix ratio was the highest,reaching 50.66%of the total secondary structure;the?-turn angle ratio was the smallest,accounting for 6.91%of the total secondary structure;and the extended chain accounted for 14.80%of the total secondary structure;Random curl accounts for 27.63%of the total secondary structure.The Cep I and Rpf F genes of strain JK-SH007 are closest to strain B.stabilis LR025743 and strain B.pyrrocinia CP011504,respectively,with homology of 100%and 97.49%,respectively,and sequence similarity of 94%and 97.49%,respectively;In addition,the amino acid sequences encoded by the Cep I and Rpf F genes of JK-SH007 strain have been replaced with 9 and 7 amino acids,respectively,and the identity of the two is 91%and 93%,respectively.4.By constructing QS over-expressing strains,and comparing JK-SH007 wild strains and overexpression strains,it was found that the QS system is regulating the growth,biofilm formation,and endogenous colonization capacity of JK-SH007 strains.Compared with wild strains,the overexpression strains had higher endogenous colonization ability in the roots and stems of poplars,indicating that the QS system played a very important role in the colonization of poplar roots and stems.The above results confirm the correlation between the JK-SH007 quorum sensing system and its colonization in poplar,which is conducive to revealing the function of the quorum-sensing signal synthase gene of the strain,and also provides a new way to improve the endogenous colonization ability of the strain in plants,and provides a theoretical basis for subsequent practical applications.