The Structural And Functional Research Of TetR Family Transcriptional Regulator PhIH From Pseudomonas Fluorescens

Pseudomonas fluorescens is a Gram-negative bacterium that is a typical symbio-tic bacterium of beneficial plants.It produces a number of secondary metabolites,such as 2,4-diacetylphlophone(2,4-DAPG),hydrogen cyanide and iron carriers,which enhance Pseudomonas fluorescens Competitive advantage with other soil microorga-nisms.Among them,the antibiotic 2,4-DAPG proved to be a key factor in the inhibi-tion of plant diseases by Pseudomonas fluorescens.2,4-DAPG is a small molecule compound that is the source of many biological control of Pseudomonas fluorescens.Its synthetic gene cluster phl contains eight genes,named phlACBDEFGH,of which TetR family transcriptional regulator(TFR)PhlH has recently shown negative feedback regulation with 2,4-DAPG biosynthesis.The phlG gene encodes a C-C bond hydrolase that specifically degrades 2,4-DAPG to the less toxic monoacetylphlorog-lucinol(MAPG).PhlH can regulate the expression of phIG gene by detecting the con-centration of 2,4-DAPG,and dynamically release the potential metabolic burden of 2,4-DAPG.It provides a selection advantage for Pseudomonas fluorescens and has a competitive rhizosphere.Biological control function.Through sequence alig nment,we foun d that PhlH has low homology to other protein sequences and its structure is unknown.In this study,apo-PhlH crystal struc-ture with a resolution of 2.4 A was obtained by X-ray diffraction.Analysis of the apo-PhlH structure,we can see that the entire protein structure is in an unstable state,especially the area responsible for allosteric.In some of the loops,some amino acid residues are missing,the loop between a6 and a7,and the loop between a8 and a9.The reason for the deletion is that the region is in a flexible state,so that the electron density is not captured,thus Some amino acid residues were not completely construc-ted.In addition,the crystal structure of the PhlH-2,4-DAPG complex with a resolution of 2.1 A was obtained,and the PhlH-2,4-DAPG structure was in a low-energy stable-state.By comparing the DNA-binding domain(DBD)of apo-PhlH and PhlH-2,4-DAPG structures,we found that the PhlH-2,4-DAPG structure produced a large amount of DBD compared to the apo-PhlH structure.The displacement is increased by a distance of 34 A between two ?3s in the original apo-PhlH structure,and an increase of 12 A to 46 A forms a distance that is not easily combined with the DNA major groove.EMSA and ITC experiments reveal changes in the ability of specific amino acid residues to bind to ligands or DNA,where the seven amino acids F173,L85,L186,V190,T123,L177 and E132 are critical for the binding of 2,4-DAPG.H76 and K144 form a hydrogen bond to control the entry of 2,4-DAPG,which plays a role in gating.These results provide evidence for the possible presence of TFRs and help to understand the operon activation of molecular basis and conformational changes induced by ligand binding.