Characterization of a brown pigment from Bacillus subtilis cultures

Abstract

Bacillus subtilis cells produce brown pigments upon sporulation in liquid or solid medium, a phenotypic trait often made use of in genetic studies. One of five brown pigments produced in a defined liquid medium, designated pigment T1, has been purified to homogeneity by gel permeation chromatography, butanol extraction, and absorption chromatography on microcrystalline cellulose and silica gel. The molecular weight of pigment T1 was estimated to be 3500. The pigment has a featureless visible absorption spectrum (absorbance increases logarithmically with decreasing wavelength) and a shoulder in the ultraviolet absorption spectrum at 260 nm. Pigment T1 exhibited a yellow fluorescence; the excitation maximum was 292 nm, and the emission maximum was 367 nm. The compound was freely soluble in water, methanol, and dimethylsulfoxide, contained 1 mol of carbohydrate per 3500 g but no Mn2+ and no amino acids which could be released by acid hydrolysis. Elemental analysis of pigment T1 gave the following: C, 44.02%; H, 6.40%; N, 10.35%; O, 36.49%; and S, 2.74%, which corresponded to a simplest formula of C43H75N9O27S1. Although it had been suggested previously that the brown pigments of B. subtilis were melanins, it was concluded from the data given here that pigment T1 is not a melanin.