Phenotypic and GWAS analyses demonstrate that VNBI isolates have a greater ability to melanize than VNBII isolates and identify BZP4-deficient isolates as having reduced melanization capacity. (A) Isolates were provided with L-DOPA, and colony brightness was assayed; isolates with the lowest brightness are the most melanized. Clinical isolates are shown in red, environmental isolates are shown in blue, control/unknown isolates are shown in black, and isolates described in B are underscored with numbered circles. VNBI clinical isolates were significantly more melanized than VNBII clinical isolates (P < 0.00017), and VNBI environmental isolates were significantly more melanized than VNBI clinical isolates (P < 0.00047). For the latter comparison, the least melanized (brightness ≥0.6) samples were excluded to prevent an effect of sampling bias. (B) GWAS analysis identified loss-of-function mutations in BZP4 as being associated with a lack of melanization. The four isolates with BZP4 loss-of-function mutations are shown here in the L-DOPA assay, along with the positive control H99 and the negative control lac1Δ. (C) A strong correlation of melanization was present between replicates. Isolates shown in B are indicated with numbered circles.
