Identification of a Pantoea biosynthetic cluster that directs the synthesis of an antimicrobial natural product

Abstract

Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.

Figure 1. The composition and organization of the 8.2-1 antibiotic biosynthetic cluster in P. ananatis BRT175 (Accession: ASJH00000000)

. The insertion sites of the mini-Tn5 transposon for mutants generated by transposon mutagenesis are indicated with lines within the cluster. Homologous biosynthetic gene clusters of P. syringae pv. maculicola ES4326 (11.6 kb) (Accessions: AEAK01000141, AEAK01000142), P. fluorescens WH6 (11.6 kb) (Accession: AEAZ01000041), and P. chlororaphis PCL1391 (7.5 kb) (Accession: DQ367408) are shown. A gene cluster in P. agglomerans Eh318 (10kb) (Accession: AXOF00000000) having several homologous genes is shown. Gene colours indicate homology between the biosynthetic clusters. Cluster in P. syringae pv. maculicola ES4326 overlaps two contigs.