Isolation and Characterization of the SerotypegCarbohydrate Moiety from an Enzyme Lysate ofStreptococcus mutans6715 Cell Walls

Abstract

The serotype‐specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell‐wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo‐N‐acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C‐25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G‐100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A‐25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re‐chromatographed on a Bio‐Gel P‐100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti‐serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti‐serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α‐linked galactose‐glucose terminal linkage.