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SUMMARY: The ribonucleic acid fraction of Micrococcus pyogenes var. aureus (strain Duncan), separated by the method of Schmidt & Thannhauser (1945), contains organic phosphate in excess of that which can be accounted for by a poly- nucleotide nucleic acid structure. This 'excess phosphate' can be separated from the other phospho-compounds after fractional extraction of M. pyogenes in dilute acid or in dilute alkali. Four polyolphosphate compounds occur in the extracts. The main components are a-and /3-glycerophosphate, the two other polyolphosphates (giving water-insoluble barium salts ; separable chromatographically) occurring in smaller amount. The yields of glycerophosphate show that at least 75 yo of the 'excess phosphate ' is present in an easily hydrolysed glycerophospho-compound which may also be the origin of the unidentified polyolphosphates. The glycerophospho-com- pound of M. pyogenes accounts for more than one-quarter of the total organic phosphate of the organism. The presence of 'excess phosphate' in a number of Gram-positive organisms suggests that a glycerophospho-compound similar to that of M. pyogenes may be of general occurrence in Gram-positive bacteria and in yeasts. In the course of checking a spectrophotometric method of estimating nucleic acid in intact cell suspensions (Mitchell, 1950a) against an adaptation of the method of Schmidt & Thannhauser (1945) developed by Stephenson & Moyle (1949), it was found that although the two methods were in substantial agreement when applied to Escherichia coli (strain H), they did not agree when applied to Micrococcus pyogenes var. aureus (strain Duncan). Comparison of the light absorption at a wave-length of 260 mp. with the organic phosphate content of the ribonucleic acid (RNA) fraction of the Schmidt & Thannhauser method indicated that in M. pyogenes there was an excess of organic phosphate amounting to more than 30 yo of that which could be accounted for by a polynucleotide RNA structure (Mitchell, 1950a). The occurrence of 'excess phosphate' in the RNA fractions of a number of micro-organisms showed a strong correlation with the capacity for retaining Gram's stain (Mitchell & Moyle, 1950), and it was observed that the amount of 'excess phosphate' in M. pyogenes was decreased during growth in the presence of penicillin (Mitchell, 1950b; Mitchell & Moyle, 1951). It was desirable to begin a study of the origin of the 'excess phosphate' of the RNA fraction of Gram-positive bacteria not only to facilitate the elimina- tion of errors in the estimation of RNA, but also to account for the occurrence of a large fraction of the total organic phosphate of these organisms. The present paper describes work on the separation and characterization of fragments of a phospho-compound containing the major part of the 'excess phosphate' of the RNA fraction of M. pyogenes var. aureus (strain Duncan), cultivated as described by Mitchell (1950 a).

Isolation of Hydrolytic Products of a Glycerophospho-compound from Micrococcus pyogenes