Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6

Abstract

Two melanin-overproducing Pseudomonas putida F6 mutants were generated using transposon (Tn5) mutagenesis. Mutants were disrupted in a transcriptional regulator (TR) and a homogentisate 1,2-dioxygenase (HDO) gene. Colonies of mutant F6-TR overproduced a black pigment on solid medium. The same mutant (F6-TR) had a 3.7-fold higher tyrosinase activity compared with the wild-type strain when induced with ferulic acid. However in tyrosine uptake assays whole cells of the mutant strain F6-TR consumed eight times less tyrosine compared with the wild-type strain. Mutant F6-HDO produced a diffusible red pigment into the growth medium. Pigment production by mutant F6-HDO is sixfold higher than the wild-type strain. The biomass yield of mutant F6-HDO grown on tyrosine as the sole source of carbon and energy was 1.2-fold lower than the wild-type strain. While the growth of the wild-type strain was completely inhibited by 5 min of exposure to UV light (254 nm) both mutant strains showed survival rates gt 30%. Mutant F6-HDO was able to tolerate higher concentrations of hydrogen peroxide (H(2)O(2)) exhibiting 1.5 times smaller zones of inhibition at 10 mM H(2)O(2) compared with mutant F6-TR and the wild-type strain. The pigments produced by all strains were purified and confirmed to be melanins.