Use of PCR-Targeted Mutagenesis To Disrupt Production of Fusaricidin-Type Antifungal Antibiotics in Paenibacillus polymyxa

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Abstract
Paenibacillus polymyxa (formerly Bacillus polymyxa ) PKB1 has been identified as a potential agent for biocontrol of blackleg disease of canola, caused by the pathogenic fungus Leptosphaeria maculans . The factors presumed to contribute to disease suppression by strain PKB1 include the production of fusaricidin-type antifungal metabolites that appear around the onset of bacterial sporulation. The fusaricidins are a family of lipopeptide antibiotics consisting of a β-hydroxy fatty acid linked to a cyclic hexapeptide. Using a reverse genetic approach based on conserved motifs of nonribosomal peptide synthetases, a DNA fragment that appears to encode the first two modules of the putative fusaricidin synthetase ( fusA ) was isolated from PKB1. To confirm the involvement of fusA in production of fusaricidins, a modified PCR targeting mutagenesis protocol was developed to create a fusA mutation in PKB1. A DNA fragment internal to fusA was replaced by a gene disruption cassette containing two antibiotic resistance genes for independent selection of apramycin resistance in Escherichia coli and chloramphenicol resistance in P. polymyxa . Inclusion of an oriT site in the disruption cassette allowed efficient transfer of the inactivated fusA allele to P. polymyxa by intergeneric conjugation. Targeted disruption of fusA led to the complete loss of antifungal activity against L. maculans , suggesting that fusA plays an essential role in the nonribosomal synthesis of fusaricidins.