Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440

摘要: A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed optimized for low basal expression. Mannitol, arabitol, glucitol-inducible gene expression demonstrated with putida eGFP as reporter gene. The applied functional characterization of PmtlE. Identification DNA binding site MtlR achieved by in vivo measurement wild mutants thereof. Moreover, purified detailed vitro investigations using electrophoretic mobility shift assays DNaseI footprinting experiments. obtained data suggest that binds to a dimer. proposed is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. transcription activation mechanism includes two sites different affinities, strong upstream weaker downstream site. presence weak shown be necessary sustain mannitol-inducibility Two possible functions are discussed; effector might stabilize second monomer half or promote inducing conformational change regulator influences contact RNA polymerase.