Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain

Vibrio anguillarum 531A, isolated from a diseased fish in the Atlantic Ocean, is a mixture composed of about 95 and 5% of highly pigmented cells (strain 531Ad) and cells with normal levels of pigmentation (strain 531Ac), respectively. Analysis of the V . anguillarum 531Ad DNA region encompassing genes involved in the tyrosine metabolism showed a 410-bp duplication within the hmgA gene that results in a frameshift and early termination of translation of the homogentisate 1,2-dioxygenase. We hypothesized that this mutation results in accumulation of homogentisate that is oxidized and polymerized to produce pyomelanin. Introduction in E . coli of recombinant clones carrying the V . anguillarum hppD (4-hydroxyphenylpyruvate-dioxygenase), and a mutated hmgA produced brown colored colonies. Complementation with a recombinant clone harboring hmgA restored the original color to the colonies confirming that in the absence of homogentisate 1,2-dioxygenase the intermediary in tyrosine catabolism homogentisate accumulates and undergoes nonenzymatic oxidation and polymerization resulting in high amounts of the brown pigment. Whole-genome sequence analysis showed that V . anguillarum 531 Ac and 531Ad differ in the hmgA gene mutation and 23 mutations, most of which locate to intergenic regions and insertion sequences.