title: Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii.
abstract: Based on genomic analysis, polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways account for biosynthesis of the majority of the secondary metabolites produced by the entomopathogenic fungus Metarhizium robertsii. To evaluate the contribution of these pathways to M. robertsii fitness and/or virulence, mutants deleted for mrpptA, the Sfp-type 4' phosphopantetheinyl transferase gene required for their activation were generated. ΔmrpptA strains were deficient in PKS and NRPS activity resulting in colonies that lacked the typical green pigment and failed to produce the nonribosomal peptides (destruxins, serinocylins, and the siderophores ferricrocin and metachelins) as well as the hybrid polyketide-peptides (NG-39x) that are all produced by the wild type (WT) M. robertsii. The ΔmrpptA colonies were also auxotrophic for lysine. Two other mutant strains were generated: ΔmraarA, in which the α-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and ΔmrsidA, in which the L-ornithine N5-oxygenase gene that is critical for hydroxamate siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of ΔmrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine biosynthesis marginally increased resistance to H2O2while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, combined loss of metachelin and ferricrocin through the inactivation of mrsidA resulted in mutants that were as hypersensitive or slightly more sensitive to H2O2, iron deprivation, and SDS, and were either identical or marginally higher in ΔmrpptA strains. In contrast to ΔmrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggests the production of one or more non-hydroxamate iron-chelating compounds. Deletion of mrpptA, mrsidA, and mraarA reduced conidium production and conidia of a GFP-tagged ΔmrpptA strain displayed a longer germination delay than WT on insect cuticles, a deficiency that was rescued by lysine supplementation. Compared with WT, ΔmrpptA strains displayed ∼19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ∼2 and ∼6-fold decreases in virulence, respectively. Deletion of mrpptA had no significant effect on growth inhibition of Bacillus cereus. Our results suggest that PKS and NRPS metabolism plays a significant role in M. robertsii virulence, depresses conidium production, and contributes marginally to resistance to oxidative stress and iron homeostasis, but has no significant antibacterial effect.
authors.affiliation: School of Integrative Plant Science - Plant Pathology and Plant-Microbe Biology Section, Cornell University, Ithaca, NY 14853, United States. Electronic address: bdd1040@gmail.com.
authors.forename: Bruno Giuliano Garisto
authors.initials: BGG
authors.lastname: Donzelli
authors.affiliation: School of Integrative Plant Science - Plant Pathology and Plant-Microbe Biology Section, Cornell University, Ithaca, NY 14853, United States.
authors.forename: B Gillian
authors.initials: BG
authors.lastname: Turgeon
authors.affiliation: USDA ARS, Robert W. Holley Center for Agriculture and Health, 538 Tower Road, Ithaca, NY 14853, United States.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Gibson
authors.affiliation: USDA ARS, Robert W. Holley Center for Agriculture and Health, 538 Tower Road, Ithaca, NY 14853, United States. Electronic address: stuart.krasnoff@ars.usda.gov.
authors.forename: Stuart B
authors.initials: SB
authors.lastname: Krasnoff
mesh_terms: D000818:Animals; D004330:Drosophila / Q000382:microbiology; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis / Q000235:genetics*; D052981:Metarhizium / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D009154:Mutation; D018384:Oxidative Stress / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D064210:Secondary Metabolism / Q000235:genetics; D013172:Spores, Fungal / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity
chemical_list: D005656:Fungal Proteins; D048630:Polyketide Synthases; D007501:Iron; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D008239:Lysine
keywords: 4′ Phosphopantetheinyl transferase; Entomopathogen; L-Ornithine N(5)-oxygenase; NRPS; PKS; Siderophores; α-Aminoadipate reductase