Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2004
Publication Date: 11/1/2004
Citation: Anderson, L.M., Stockwell, V.O., Loper, J.E. 2004. An extracellular protease of pseudomonas fluorescens a506 inactivates antibiotics of pantoea agglomerans. Phytopathology. 94:1228-1234. Interpretive Summary: Biological control is a promising approach for the management of plant diseases such as fire blight, an important disease of pear and apple. Fire blight is caused by a bacterial pathogen called Erwinia amylovora. For the past several decades, growers managed fire blight by spraying trees with antibiotics like streptomycin. Because, in many regions, the pathogen is now resistant to streptomycin, biological control is becoming increasingly important for management of fire blight. This research is part of a greater effort to develop improved biological control for fire blight. This work evaluated the production of protease, an extracellular enzyme, by the biological control agent Pseudomonas fluorescens A506, a registered biological control product for fire blight. A mutant of A506 lacking protease production was derived and characterized. The protease was shown to degrade an antibiotic produced by Pantoea agglomerans, another biological control agent used to suppress fire blight. This research represents the first step in a larger effort to develop improved biological control for fire blight. It also extends knowledge of microbial proteases and their roles in the interactions between microorganisms.
Technical Abstract: Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pa. agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, purified extracellular protease inactivated the antibiotics MccEh252 and herbicolin O, which are produced by Pa. agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. A506 inactivated MccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously-unknown role of the extracellular protease produced by P. fluorescens in microbial interactions and extend the previously recognized roles of such proteases in bacterial nutrition or virulence.