Genetic and evolutionary characterization of the Major Facilitator Superfamily transporters of the antibacterial, Pantoea Natural Product 3

Pantoea Natural Product 3 (PNP-3) is an antibiotic produced by Pantoea agglomerans that is effective against a broad range of multi-drug resistant bacteria. PNP-3 is encoded by a unique, eight-gene biosynthetic gene cluster composed of predicted enzymes (pnp3b, pnp3e–h), a regulator (pnp3d), and two Major Facilitator Superfamily transporters (pnp3a and pnp3c). To better characterize the role of the transporters, we generated pnp3a and pnp3c mutants and evaluated PNP-3 production. Disruption of pnp3a in Pantoea results in impaired growth and loss of antibiosis, suggesting a role in PNP-3 export and resistance. In contrast, pnp3c mutants display only reduced antibiotic production/export, suggesting a minor role for Pnp3c. Expression of pnp3a in susceptible Erwinia amylovora led to increased PNP-3 tolerance, while co-expression of pnp3a and pnp3e–h resulted in the production and export of PNP-3. Comparative genomic analyses identified pnp3a in 12 other Pantoea strains, eight of which carry a complete or nearly complete PNP-3 biosynthetic cluster. The four other Pantoea strains that carry pnp3a lack most of the PNP-3 cluster; however, they are PNP-3 tolerant. These results suggest Pnp3a plays an essential role in PNP-3 export and resistance in Pantoea.