Screening,validation And Functional Study Of Diguanylate Cyclase Interacting Proteins From Rhodococcus Ruber SD3

Diguanylate cyclase（DGC）regulates many important physiological processes such as the formation and breakdown of bacterial biofilm,motility,production of extracellular polysaccharide,pathogenicity,and cell morphology by regulating the concentration of intracellular c-di-GMP.Our previous studies indicated that diguanylate cyclase in R.ruber SD3 is closely associated with the organic solvent tolerance of this strain,but the detailed mechanism needs further research.To further explore the biological function of diguanylate cyclase,the present study aims at identifying the protein on the surface of R.ruber SD3 cell that can interact with diguanylate cyclase and revealing the molecular mechanism of how R.ruber SD3senses chemicals and transfers these external stimuli.This not only has important scientific value for elucidating the unique physiological and biochemical characteristics of R.ruber SD3,but plays a practical role in the field of biocaalysis and bioremediation.The main research contents and results of this paper are as follows:A proteins named DGCIP1 that might interact with DGC was obtained by screening and prediction from STRING database.The bioinformatics analysis indicted that DGCIP1 had the molecular weight of 60.8 k Da and the theoretical p I of4.96.It also has a instability index of 31.68,aliphatic index of 93.36 and a grand average of hydropathicity of-0.189,and thus it is a stable and hydrophilic protein.The protein has two transmembrane segments,and the secondary structure is mainlyα-helix,accounting for 61.52%.DGCIP1 contained CHASE3,HAMP,His KA and HATPase conserved domains Tsrp1 in R.ruber SD3 has a similarity of 94.86,68.80%,68.42%,64.97%,65.17%,63.83%and 63.79%with kinases in R.rhodochrous,R.pyridinivorans,R.gordoniae,S.bicolor,S.canus,S.cinerochromogenes and S.incarnatusThe corresponding sequence of DGCIP1 was applied for codon optimization,and DGCIP1 was heterogeneously expressed in E.coli BL21（DE3）using expression plasmid p ET-28a.The optimal expression conditions of DGCIP1 were as follows:the induction temperature of 25℃and the IPTG concentration of 0.1 m M.The recombinant with electrophoresis purity was obtained by affinity chromatography.Blue Native PAGE showed that The recombinant DGCIP1 formed trimers under natural conditions.The interaction between DGCIP1 and diguanylate cyclase DGC was analyzed using GRAMM-X and PDBe PISA.The results showed that the free energy of interaction between the two was-17.1 kcal/mol,with the interface area of 30957?².List of interaction indicated there were eight hydrogen bonds,two salt bridges and no sulfide bond and covalent bond between the two proteins.GST Pull-down experiment results also showed that there was an interaction between the two proteins.DGCIP1was found to have a Km value of 21.10μM,a Vmax value of 1.13μm/min,and a Kcat value of 0.23 s^-1by enzymatic kenetics.Sixty two common environmental pollutant compounds were docked with CHASE3 by Auto Dock.The results showed that 44 small molecules had significant hydrogen bonding forces with CHASE3.The other 18 compounds had hydrophobic interaction with CHASE3.The binding free energy of 42 small molecules and CHASE3 is less than-2 kcal/mol.The affinity between CHASE3 and 6 small molecules was tested in MST assay.The MST experiment showed that CHASE3 had affinity with the 6 small molecules.Blue Native PAGE showed that CHASE3 formed trimers.Hexamers and octamers under natural conditions.In conclusion,a protein named DGCIP1 which can interacted with diguanylate cyclase was identified in the study.The bioinformatics analysis and ATP hydrolysis activity of DGCIP1 suggest it is a histidine kinase.The interaction between CHASE3and some compounds were verified,laying a foundation for a comprehensive understanding of relationship between diguanylate cyclase mediated signaling pathway and the organic solvent tolerance of R.ruber SD3.