Our research has shown the carotenoid pigment to be found in the cell membrane of C. poinsettiae. These results suggest that the pigment is bound to protein and that protein tertiary structure is important in the binding.

The purpose of this study was to completely characterize the protein moiety in the caroteno complex in C. poinsettae, determine if the distribution and level of protein in the pigment-protein complex in membranes of the wild type and in a colorless mutant could account for the differences in the stability of the membrane, and to determine if this protein is common to other pigmented and non-pigmented organisms. Also, electron microscopy of cell membranes of C. poinsettiae which had been exposed to gold-labelled antibody against the protein moitey of the pigment-protein complex, demonstrating that the protein is randomly distributed in the membranes of both wild type and colorless mutant.

The purpose of this study was (1) to determine whether a correlation exists among the protein profiles, extracted from cell membranes of mutants belonging to five pigment cluster groups, (2) to locate the protein moiety and cartenoprotein complex in the membranes of wild type and colorless mutant (designated W-19) of C. poinsettae and to show whether there are any structural differences between cell membranes of the wild type and a colorless mutant, (3) to determine the effect of six antibiotics on cartenoid gene expression.

In a minimal medium supplemented with glucose and yeast extract, the optimum pH for the growth of C. poinsettias was found to be 7.5. The organism requires thiamine, biotin, and pantothenic acid for growth. No absolute requirement was found for any amino acid, purine or pyrimidine although an amino acid mixture was stimulatory. Casamino acids could be substituted for the synthetic amino acid mixture. Yeast extract provided an additional factor(s) necessary for maximal growth. The results suggest that the unknown factor found in yeast extract might be purified by a combination of solvent extraction, and adsorption and elution from charcoal.

Carotenoid pigments were extracted from Corynebacteriuma poinsettiae (wild type) ATCC 9682, and from 108 mutants obtained by exposure of a streptomycin resistant strain of C. poinsettiae to ultra-violet light irradiation and N-methyl- N'-nitro-N-nitrosoguanidine. The pigments were characterized by their absorption maxima, Rf-values, and partition ratios in petroleum ether and methanol. Thin layer chromatography was used to compare pigments of the wild type with those of the mutants. Possible biosynthetic pathways in carotenoid synthesis of the wild type were postulated on the basis of the observed genetic blocks. Mutants were found which suggested the existence of a linear pathway in carotenoid synthesis from the aliphatic C4 0 molecule to the bi-cyclic C50-diol. Other mutants suggested possible alternative pathways in the biosynthesis of these pigments or the presence of intermediates not detectable by thin layer chromatography.

Corynebacterium poinsettiae mutant strains blocked in carotenoid biosynthesis were obtained by treatment with the mutagen N-methyl-N1-nitro-N-nitrosoguanidine. Additional carotenoid (Crt) mutant strains were obtained from a previous study conducted in our laboratory. Fifty-nine Crt mutants affected in carotenoid biosynthesis were examined by a normal phase high performance liquid chromatography (HPLC) system. Pigment extracts of Crt mutants and C. poinsettiae wild type strains were resolved by an isocratic system with hexane:acetone:dicholoromethane, 11.35:1.73:1.00 (by vol.) as the eluting solvent. In addition to the five major peaks, twelve minor peaks were observed in the wild type C. poinsettiae strain used in this study. Crt mutant and wild type strain peak heights were measured from the individual chromatograms and the peak height data set created was analyzed using the Statistical Analysis System program to perform a cluster analysis. The cluster analysis revealed five carotenoid mutant groups. Carotenoid pigments which accumulated or were absent in each of the cluster groups are reported. Cluster group 1 mutants (CrtA) are blocked in the dehydrogenase(s) which is(are) responsible for the dehydrogenations between phytoene and lycopene. Cluster group 2 mutants (CrtB) appear to be blocked at a second dehydrogenase specific for the dehydrogenation from C.p. 470 to C.p. 496. Cluster group 3 mutants (CrtC) are blocked at a cyclization step in the pathway which involves cyclization of C.p. 496 to C.p. 470 and which may cyclize C.p. 473 to C.p. 450. The genes CrtA and CrtB map only 0.5 map units from each other while CrtA and CrtC map 2.1 map units from one another. Mutants which accumulate end products but which lack certain precursors indicate a branched pathway for pigment biosynthesis exists in this organism. Media for the formation, fusion and regeneration of C. poinsettiae protoplasts are reported and a protocol for the use of these media in genetic crosses of strains blocked in carotenoid biosynthesis is described. While isolating antibiotic resistant mutants useful in genetic analyses, novobiocin resistant mutants were observed to have a distinctly different colony pigment phenotype as compared to the wild type strain. HPLC chromatograms of a novobiocin resistant strain showed a distinctly different carotenoid pigment profile. The results provide evidence for differential gene expression in the carotenoid biosynthetic pathway when these mutants are grown in the presence of novobiocin.