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Abstract
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 and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.
Highlights
Pseudomonads thrive in various ecological niches such as soil, plants, rhizosphere, water bodies, humans, and animals
In order to construct an expression vector containing the functional mtlR/ PmtlE regulatory unit, PmtlE was fused to the reporter gene eGFP and integrated into a pBBR1MCS-2-based vector
Expression of eGFP was investigated with P. putida GN146 pJOE7771.1 with or without mannitol, arabitol, or glucitol, respectively and the obtained fluorescence data were compared to those of the well-known RhaR-RhaS/PrhaBAD [32,50] and TetR/PtetA expression systems
Summary
Pseudomonads thrive in various ecological niches such as soil, plants, rhizosphere, water bodies, humans, and animals. They possess the metabolic power to utilize a remarkably broad range of substrates, including carbohydrates, fatty acids, organic acids, alcohols, amines, amino acids as well as aromatic and aliphatic hydrocarbons [1]. The Mannitol Promoter from Pseudomonas fluorescens doi:10.1371/journal.pone.0133248.g001. The mannitol utilization genes of P. fluorescens DSM50106 are organized in an operon consisting of seven catabolic genes encoding the components for mannitol transport and conversion [2,3]. The operon components mediate transport and utilization of glucitol and arabitol (Fig 1). Translocation of mannitol, arabitol, and glucitol into the cytoplasm is mediated by an ABC transporter encoded by mtlE, mtlF, mtlG, and mtlK. Fructose and xylulose are phosphorylated by kinases MtlY and MtlZ and channeled into the intermediary metabolism
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