The genetics of antibiotic production and the role of antibiotics in biological control of Erwinia amylovora by Erwinia herbicola

Sammanfattning: Erwinia herbicola is an epiphyte of apple and pear with potential as a biocontrol agent of Erwinia amylovora, which causes fire blight. This research aimed to assess the relative role of antibiotics produced by E. herbicola, Eh318 as a mechanistic basis for biocontrol. A genomic library of Eh318 DNA was constructed in Escherichia coli and two distinct cosmids, pCPP702 and pCPP704, were identified that conferred upon E. coli the ability to produce two antibiotics inhibitory to E. amylovora. The antibiotics were distinct based on their spectra of activity, differential susceptibility to the presence of histidine and arginine and antibiotic production by marker-exchange mutants of Eh318. Transposon mutagenesis and subcloning were used to delineate the Eh318 DNA that enabled E. coli to produce the two antibiotics. The smallest clone that conferred antibacterial activity was pCPP717. Its antibiotic was named pantocin A. The Eh318 insert DNA of pCPP717 revealed three predicted genes, paaA, paaB and paaC, in a 2.7 kb region. The predicted paaA gene product is similar in sequence to a group of biosynthetic enzymes that possess a dinucleotide binding motif. PaaC was judged to encode a membrane protein. The second antibiotic was named pantocin B. Its synthesis is conferred on E. coli by DNA harbored in clone pCPP719. Between 19 kb and 20 kb of Eh318 DNA is needed for the production of pantocin B. Direct Tn5- and marker-exchange mutants of Eh318, deficient in pantocin A and/or pantocin B, were created. The mutant strains were tested for biocontrol ability in immature pear fruit in the laboratory and in apple blossoms in a controlled environment chamber. Results from both assays revealed that the marker-exchange mutant deficient in both antibiotics (Eh440) protected against fire blight less well than Eh318. The single marker-exchange mutants, Eh421 (deficient in pantocin A) and Eh439 (deficient in pantocin B), were not significantly impaired in biocontrol ability, whereas three directly induced Tn5-mutants, Eh454, Eh464 and Eh468, were less effective than Eh318. Thus, pantocins contribute to but are not solely responsible for the biological control of fire blight by E. herbicola Eh318.