Formation of indigo and related compounds from indolecarboxylic acids by aromatic acid-degrading bacteria: chromogenic reactions for cloning genes encoding dioxygenases that act on aromatic acids.

The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p-toluate-degrading strain P. putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin. A mechanism by which these products could be formed spontaneously following dioxygenase-catalyzed dihydroxylation of the indolecarboxylates is proposed. Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase. Dioxygenase gene-carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate.