The Action of Hot Formamide on Bacterial Cell Walls

The cell walls of Corynebacterium tritici contain much carbohydrate and their mucopeptide contains diaminobutyric acid instead of lysine or diaminopimelic acid. They are resistant to lysozyme. 2 . The residue after extraction with hot formamide contains only about 10% less carbohydrate but is attacked by lysozyme. Lysozyme also slowly attacks cell walls treated with fluorodinitrobenzene and more rapidly cell walls that have been N-acetylated. 3 . All these processes block the free y-amino groups of diaminobutyric acid present in the untreated cell wall. Hot formamide introduces formyl groups, as shown by its ability to make formylglycine and diformyl-lysine under the same conditions. 4 . N-Formyl groups are also introduced into the cell walls of Micrococcus ly8odeikticu8 by hot formamide, but this change increases only slightly their already great sensitivity to lysozyme. N-Acetylation also increases sensitivity to lysozyme. Fuller (1938) introduced the use of hot formamide for the extraction of antigens from dried bacteria. Recently the technique has been applied to isolated cell walls to remove polysaccharides from the struc- tural mucopeptide component (McCarty, 1960; MacLennan, 1961; Heymann, Zeleznick & Man- niello, 1961; Heymann, Manniello & Barkulis, 1963; Perkins, 1963). It has also been shown that treat- ment with hot formamide of cell walls, which were resistant to the action of lysozyme, yielded muco- peptide residues that the enzyme would attack (Krause & McCarty, 1961; Allsop & Work, 1963; Heymann, Manniello & Barkulis, 1964). The last- mentioned authors found that formamide at 1700 introduced O-formyl groups into the cell walls of streptococci and that these groups had to be re- moved before lysozyme would act on the muco- peptide substrate. The connexion between sensi- tivity to lysozyme and the presence of O-acyl groups in the cell walls of some species is well known (Abrams, 1958; Brumfltt, Wardlaw & Park, 1958).