Genomic insights into the potent antifungal activity of B. ambifaria T16

Burkholderia ambifaria T16 is a bacterial strain isolated from the rhizosphere of barley plants, which showed a potent antifungal activity against several Fusarium species. In this work, the construction of a mini-Tn5 insertional library in this strain allowed us the identification of several genes involved in the antifungal activity. In particular, several mutants with mini-Tn5 insertions located in a biosynthetic gene cluster (BGC) involved in the production of cyclic glycolipopeptides known as occidiofungins (Ocfs) were completely unable to inhibit the in vitro growth of F. oxysporum. Tandem mass spectrometry analysis of lipopeptide extracts obtained from culture supernatants of the wild-type strain allowed the identification of eight Ocfs structural variants, that were not detected in extracts of the mutants with insertions in the ocf gene cluster. These results indicate that the antifungal activity of B. ambifaria T16 mainly relies on Ocfs production. Moreover, an insertion in a BGC predicted to be involved in the biosynthesis of a membrane-associated lipopeptide designated AFC-BC11 reduced the antifungal activity of B. ambifaria T16 by approximately 50%. Accordingly, membrane extracts from the insertional mutant in the afc-BC11 cluster showed a marked reduction in the antifungal activity against several fungi. In contrast, the antifungal activity of the membrane fraction of mutants unable to produce Ocfs was not affected. Furthermore, a detailed bioinformatic analysis of the proteins encoded by the ocf and afc-BC11 gene clusters revealed that the biosynthesis of these lipopeptides would be initiated by fatty acyl-AMP ligase (FAAL) domain containing enzymes and continued by a hybrid non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) or a type III PKS, respectively. These results provide new insights into lipopeptide biosynthesis, which may be valuable in the field of novel antifungal compound design.