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Bilirubin incorporation into spiroplasma membranes and methylation of spiroplasmal DNA.

Spiroplasma floricola (BNR-1), Spiroplasma sp. MQ-1 and S. apis (B-31) grown in media containing horse serum exhibited intense yellow pigmentation. Yellow pigments were not observed in S. citri (R8A2) and Spiroplasma sp. strains BC-3 and PPS-1 grown in the same medium. The reddish-yellow pigment showed up in lipid extracts of both spiroplasma membranes and horse serum. It exhibited the typical features of bilirubin: specific absorption spectrum from 390 to 500 nm with a peak at 453 nm, and a characteristic sequence of color changes on addition of HNO3 to its solution in chloroform. The pigment comigrated with commercial bilirubin from bull gall and stained greenish blue when subjected to mild oxidation by iodine. S. floricola contained 5.4 micrograms bilirubin/mg cell protein or 9.7 micrograms bilirubin/mg membrane protein. High-performance liquid chromatography (HPLC) showed the presence of significant amounts of 5-methylcytosine and very little 6-methyladenine in the DNA of S. floricola, S. apis and Spiroplasma sp. strains PPS-1 and MQ-1. S. citri and Spiroplasma sp. strain BC-3 contained 6-methyladenine and very little, if any, 5-methylcytosine. The methylated cytosine residues in Spiroplasma sp. MQ-1 were almost exclusively located in the sequence CpG, as in eukaryotes.