GREEN FLUORESCENT PIGMENT ACCUMULATED BY A MUTANT OF CELLVIBRIO GILVUS

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Abstract
A mutant of Cellvibrio gilvus, designated straln-139A, liberated a green fluorescent pigment into the surrounding culture medium. A study of the factors which affected the accumulation of this pigment led to the development of a chemically defined medium which supported maximal pigment accumulation in aerated liquid cultures. D-Glucose, glyclne or L-serine, L-phenylalanine, L-proline, and L-lysine comprised the organic components of this medium. The visible absorption spectrum of the pigment showed a maximal band at 400 m[mu] (pH 7.0). A difference spectrum between reduced and oxidized pigment showed loss of the band at 400 m[mu] upon oxidation. However, a methanol-extractable flavine like compound occurred in the wild strain but not in the mutant. Ferric ions added to the defined medium stimulated growth, with a concomitant reduction of pigment accumulation. Pigment was formed at a maximal rate during the stationary growth phase, and the highest yield was obtained by 18 hr. Organic solvents did not extract the pigment from water solutions. One and sometimes two, compounds absorbing at 400 m[mu] could be eluted by ion-exchange chromatography on Cellex-P (H+), which was used to separate the pigment from other components in the culture supernatants so that the radioactivity of the pigment could be measured. The mutant synthesized C14-labeled pigment from D-glucose-U-C14 andfrom each of four amino-acids (glycine-1-C14, L-phenylalanine-U-C14, L-proline-U-C14 and L-lysine-U-C14. [DELTA] -Amino-levulenic-acid-4-C14 did not contribute C14 to the pigment.