Lack of Murein in a Formamide-Insoluble Fraction from the Stable L-Form of Streptococcus faecium
Journal of Bacteriology
Formamide-insoluble material was isolated from the L-form of Streptococcus faecium strain F24, but this material was not murein. Group A streptococcal L-forms have been shown by Edwards and Panos (2) to contain the nucleotide precursors for murein (mucopeptide) synthesis. Although unconfirmed chemically, the nucleotide precursors for murein were found by paper chromatography (King and Altenbern, unpublished data) in extracts from the stable L-form of Streptococcus faecium strain F24 (ATCC
... ain F24 (ATCC 19635). James, Hill, and Maxted (4) reported the isolation of murein from L-forms of S. pyogenes by a method utilizing hot formamide. This report described the chemical analyses of formamide-insoluble residues isolated from the S. faecium L-form. Our results differ from those of James et al. and suggest that murein is lacking in the enterococcal L-form. Our methods were adapted from those of James et al. (4). The organisms were grown overnight at 37 C from a 1% (v/v) inoculum added to fresh Brain Heart Infusion broth containing 0.43 M NH4Cl and 0.5% additional glucose. The L-forms were harvested by centrifugation for 15 min at 5,860 X g and washed three times with physiological saline containing enough deoxyribonuclease to disperse clumps of the organisms. The saline-washed organisms were washed three times with distilled water by centrifugation for 30 min at 100,500 X g. The final pellet was lyophilized and weighed. After overnight chloroform-methanol (2:1) extraction, the solubilized lipids were separated from insoluble material by centrifugation, dried, and weighed. The chloroform-methanol-insoluble material was dried, weighed, and treated overnight at 37 C with phosphate-buffered saline (PBS, pH 7.5) containing trypsin (0.1 mg/ml). The trypsin-treated material was centrifuged for 10 min at 12,100 X g and washed three times with PBS and three times with distilled water. After the pellet was lyophilized and weighed, the material was treated twice at 170 C with formamide. After each treatment, the insoluble material was collected by centriugation for 30 min at 34,800 X g. For final gravimetric analysis, the formamide-insoluble material was washed three times with PBS and three times with distilled water, resuspended in distilled water, and transferred quantitatively to tared weighing vessels for gravimetric analysis.