THE MANNITOL UTILIZATION GENES OF PSEUDOMONAS-FLUORESCENS ARE REGULATED BY AN ACTIVATOR - CLONING, NUCLEOTIDE-SEQUENCE AND EXPRESSION OF THE MTLR GENE

A plasmid with the galK gene under control of the promoter of the mann itol utilization genes (mtl) from Pseudomonas fluorescens DSM 50106 wa s constructed to isolate the mtl regulatory gene. An Escherichia coli galK(-) mtl(-) strain with this plasmid was used to screen a genomic l ibrary of P. fluorescens for the presence of the regulatory gene by pl ating on McConkey agar plates supplemented with galactose and mannitol . Clones carrying the regulatory gene were isolated and by complementi on assays, deletion analysis and DNA sequencing an open reading frame (mtlR) of 906 nt identified encoding the regulator. The deduced protei n MtlR with a calculated molecular mass of 34.7 kDa showed a low overa ll similarity to several other regulatory proteins of the XylS/AraC fa mily. When mtlR was cloned and expressed in E. coli, the protein was p roduced as inclusion bodies. Complete denaturation followed by subsequ ent slow refolding led to low amounts of active protein. The activity was shown in gel mobility shift assays by binding of MtlR to a DNA fra gment containing the promoter/operator region of the P. fluorescens mt l genes. (C) 1998 Elsevier Science B.V. All rights reserved.