The goal of this work was to determine the chemical nature of the red pigment produced by Streptococcus agalactiae, which has been thought to be a carotene. We extracted the pigment with 0.1 M KOH and purified it by column chromatography on Sephadex LH. Data from elemental analysis and mass and nuclear magnetic resonance spectra lead us to propose the structure to be that of a new ornithine rhamno-polyene with 12 conjugated double bonds, to which we have assigned the trivial name granadaene.Streptococcus agalactiae (group B streptococcus [GBS]) is a hemolytic streptococcus which is the most important bacterium causing life-threatening infections in neonates (7). Production of a red pigment was recognized early in GBS (11), and pigment detection is a popular method for identifying GBS today (5). It was suggested (8) and is accepted that the GBS pigment is a carotene (3, 6), based on its UV-visible spectrum. Surprisingly, no further data support this, and genes coding for carotene biosynthesis are not present in GBS (7). Our data show that the GBS pigment is not a carotene but an ornithine glycopolyene, and we think that this is the first report of a glycopolyene pigment and of a polyene pigment among gram-positive bacteria.(This work was presented in part at the 7th ASM Conference on Streptococcal Genetics, Saint Malo, France, June 2006.) S. agalactiae ATCCC 12386 was grown in Erlenmeyer flasks containing Granada medium without agar (9) and incubated at 36°C in a shaker until the broth became deeply red (48 to 72 h). Cells harvested by centrifugation were washed with 0.15 M NaCl, and lipids were extracted with chloroform-methanol (2:1, vol/vol). Afterwards, pigment was extracted with KOH 0.1 M and purified as follows: (i) pigment was precipitated from the extract with methanol-1 M HCl (1:1 vol/vol); (ii) the red precipitate was washed with dimethyl sulfoxide (DMSO) and dissolved in DMSO-0.1% trifluoroacetic acid (TFA); (iii) this solution was kept in an open flask in a desiccator together with a crystallizer containing ammonium hydroxide for several hours, which results in pigment precipitation; (iv) this pigment was redissolved in DMSO-0.1% TFA and chromatographed on a column (950 by 23.5 mm) of Sephadex LH-20 (Amersham Biosciences, Denmark) which was eluted with DMSO-0.1% TFA, and the eluate peak containing the pigment (absorption at 525 nm with minimum absorption at 280 nm) was collected; and (v) pigment was reprecipitated as in step iii. For elemental analysis, pyridine/acetic anhydride acetylation, mass spectrometry (MS), and spectroscopy, the pigment was washed with water and lyophilized. For UV-visible spectroscopy, the pigment was dissolved in DMSO-0.1% TFA. For nuclear magnetic resonance (NMR) (300 MHz for The structure assigned to the GBS pigment is shown in Fig. 1. MS showed an M ϩ H ion at m/z 677.3769, and a nominal mass of 676 is assigned. Elemental analysis showed 3.8% nitrogen and that assigns two N atoms (in accordance with the nitrogen rule). The exact molecular mass and the presence of two N a...