Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain

The root-colonizing bacterium Pseudomonas fluorescens BL915 protects a variety of seedlings from damping-off disease caused by the fungal pathogen Rhizoctonia solani. Spontaneous pleiotropic mutants of P. fluorescens strain BL915 which fail to synthesize antifungal factors such as chitinase, cyanide, and pyrrolnitrin and exhibit altered colony morphology were isolated. Such mutants fail to inhibit the growth of R. solani in vitro, and their biological control capability is sharply reduced. We characterized a genomic DNA fragment from strain BL915 which, when introduced into these pleiotropic mutants, restored the lost functions, the wild-type colony morphology, and bio-control activity. DNA sequence analysis of the genomic fragment revealed the presence of genes homologous to those of numerous bacterial global regulatory systems and identified a cluster of genes identical in organization to the Escherichia coli gene cluster consisting of uvrY, uvrC, pgsA, and glyW. Coordinate biosynthesis of multiple antifungal products in some heterologous Pseudomonas strains in response to the introduction of the strain BL915 genomic fragment confirmed the regulatory nature of sequences contained on this fragment. Further genetic analysis indicated a gene homologous to response regulators of bacterial two-component systems was sufficient to complement the pleiotropic mutants and to activate antifungal genes in heterologous strains. Marker exchange of a truncated version of this gene into the P. fluorescens BL915 chromosome generated pleiotropic mutants indistinguishable from the original spontaneous mutants. Cloning and sequencing of the response regulator gene from several spontaneous mutants allowed identification of various nucleotide changes associated with the gene in such mutants.