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 mannitol utilization genes ( mtl) from Pseudomonas fluorescens DSM 50106 was constructed to isolate the mtl regulatory gene. An Escherichia coli galK − mtl − strain with this plasmid was used to screen a genomic library of P. fluorescens for the presence of the regulatory gene by plating on McConkey agar plates supplemented with galactose and mannitol. Clones carrying the regulatory gene were isolated and by complemention assays, deletion analysis and DNA sequencing an open reading frame ( mtlR) of 906 nt identified encoding the regulator. The deduced protein MtlR with a calculated molecular mass of 34.7 kDa showed a low overall similarity to several other regulatory proteins of the XylS/AraC family. When mtlR was cloned and expressed in E. coli, the protein was produced as inclusion bodies. Complete denaturation followed by subsequent 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 fragment containing the promoter/operator region of the P. fluorescens mtl genes.