Fig 1.
Detoxification pathway of 2-benzoxazolinone (BOA), and structure of a compound similar to BOA, 2-oxindole.
(A) The Fusarium verticillioides pathway for metabolic detoxification of BOA into N-(2-hydroxyphenyl)malonamic acid. The FDB1 locus facilitates hydrolysis of BOA to produce 2-aminophenol (2AP), while the FDB2 locus facilitates modification of 2AP by addition of a malonyl group to form HPMA. (B) 2-Oxindole, a γ-lactam compound structurally similar to BOA.
Table 1.
Strains used and created in this study.
Fig 2.
Gene expression analysis of F. verticillioides exposed to BOA.
Microarray expression values for selected genes of F. verticillioides grown for two hours in PDB supplemented with either ethanol alone (blue) or BOA dissolved in ethanol (red). Gene expression values are shown for the proposed FDB1 cluster (FVEG_08287 to FVEG_08295) and FDB2 cluster (FVEG_12625 to FVEG_12641) as well as for genes flanking each end of the clusters. See S1 Table for more details on the genes and their fold change in expression.
Table 2.
Genes of the FDB1 cluster.
Fig 3.
Complementation assay of the F. verticillioides fdb1 mutant with clones derived from transposon mutagenesis of cosmid F5D9.
(A) Chromosome 10 genes FVEG_08287 –FVEG_08295 (black block arrows) from the FDB1 gene cluster are shown in their transcriptional orientations. Cosmid clone F5D9 (long, thin black arrow) includes full-length copies of these genes except for FVEG_08287, which is truncated. Green and red triangles indicate the positions of transposon insertions in 20 different F5D9-derived clones following mutagenesis. Green indicates transposon insertions that did not affect complementation of the fdb1 mutant, whereas red indicates insertions that affected complementation. (B) Growth phenotypes of strain AEG 74-A4-3 transformed separately with clone F5D9 and 20 F5D9-derived clones on BOA medium (0.9 mg ml-1). Transformation of strain AEG 74-A4-3 with clone F5D9 and most F5D9-derived clones (green triangles) restored wild-type growth in the presence of BOA. Four F5D9-derived clones (red triangles; insertions 244, 264, 260 and 375) did not restore wild-type growth in the presence of BOA. The four latter transformants were also not able to metabolize BOA.
Fig 4.
(A) FVEG_08291 (MBL1) restriction map of the native and deletion alleles and Southern hybridization analysis of transformants showing the banding patterns of wild-type strain FRC M-3125 and the transformants having either homologous integration and deletion of the ORF or ectopic integration. Complementation of one of the deletion transformants is also shown (Δ08291–36::C2). Genomic DNA from all strains was digested with EcoRI (E). Wild type had a ~3.5 kb fragment whereas the deletion allele was 1.1 kb. The flank of the gene was used as probe as shown. The blot was cropped to show only the lanes of interest. (B) Growth phenotypes on BOA-amended agar of wild-type FRC M-3125 and Δmbl1 mutants targeting FVEG_08291. Mutants Δ08291–33 and Δ08291–36 were unable to grow on BOA (0.9 mg ml-1), and add-back of the gene (Δ08291–36::C2) complemented the deletion phenotype.
Fig 5.
HPLC analysis of BOA detoxification into HPMA by deletion mutants.
Chromatograms of (A) BOA and (B) HPMA standards (retention times of 10.9 and 6.69 min, respectively) in comparison to PDB amended with BOA that was either (C) left uninoculated (PDB+BOA) or was inoculated with (D) wild-type FRC M-3125 or the deletion mutants of (E) FVEG_08289 (AMD1), (F) FVEG_08290 (DLH1), or (G-H) FVEG_08291 (MBL1; two different deletion mutants). Wild type, Δamd1, and Δdlh1 were all able to fully metabolize BOA to HPMA. Two Δmbl1 mutants, Δ08291–33 and Δ08291–36, were unable to metabolize BOA. (I) Add-back of MBL1 (Δ08291–36::C2) complemented the deletion.
Fig 6.
Growth curve analysis of deletion strains in comparison to wild type.
Strains were monitored for 100 h in PDB amended with BOA (0.5 mg ml-1). OD600 measurements recorded every 2 h were plotted (mean ± standard deviation). For this experiment BOA was dissolved in DMSO, and a DMSO-only control for wild-type strain FRC M-3125 is shown (grey line). FRC M-3125 in PDB with BOA is shown with a black line. The Δamd1 strain (Δ08289–2) is the orange line with triangles. The Δdlh1 mutant (Δ08290–11) is the blue line with squares. The Δmbl1 mutants, Δ08291–33 and Δ08291–36, are the brown line with circle markers and red line with × markers, respectively. The Δmbl1::MBL1 complemented strain (Δ08291–36::C2) is the green line with × markers. Experiment was conducted twice with similar results. Data from one experiment is presented.
Fig 7.
Protein based phylogeny of FVEG_08290 (DLH1) and FVEG_12625 (DLH2).
The PhyML cladogram is shown with bootstrap values (200 replications) indicated for branches having ≥80% support. Bayesian posterior probabilities are also indicated for those branches. Zymoseptoria tritici XP_003850758 was the designated outgroup.
Fig 8.
Protein based phylogeny of FVEG_08291 (MBL1) and FVEG_12637 (MBL2).
The PhyML cladogram is shown with bootstrap values (200 replications) indicated for branches having ≥80% support. Bayesian posterior probabilities are also indicated for those branches. Zymoseptoria tritici XP_003854792 was the designated outgroup.
Table 3.
Fusarium genomes assessed by BLASTn for presence of the FDB1 and FDB2 clusters.
Fig 9.
Conserved synteny of the FDB1 cluster homologs in F. verticillioides and C. graminicola, and the FDB2 cluster homologs in F. verticillioides and A. kawachii.
The genes are color coded to show orthologs/paralogs across the clusters. As the primary genes discussed in this paper, AMD1/AMD2, DLH1/DLH2, MBL1/MBL2, and NAT1 are noted. The blue arrows depict the predicted horizontal transfers of the FDB1 cluster from Fusarium to Colletotrichum and the FDB2 cluster from Fusarium to Aspergillus. Based on the synteny comparisons, an unannotated potential pseudogene for an amino acid transporter is noted with an asterisk in the A. kawachii cluster.
Fig 10.
Hydrolysis and degradation of lactam xenobiotics.
(A) General representation of the hydrolytic cleavage of a lactam moiety, in this case a four-atom β-lactam ring. (B) The hydrolysis of BOA by Mbl1 presumably involves a similar cleavage of the lactam moiety, resulting in production of an intermediate that is further processed to 2-aminophenol presumably by Dlh1 and/or Dlh2 encoded by the FDB1 and FDB2 gene cluster, respectively. (C) The lactam-containing 2-oxindole was metabolized by all strains in this study, including the Δmbl1 mutants, thus indicating Mbl1 is not needed for the hydrolysis and degradation of this compound.