Purification and quantitative chemical analysis of cell wall peptidoglycans of Leptotrichia buccalis

Peptidoglycans of Leptotrichia buccalis ATCC 14201 and ATCC 19616 were isolated by extraction with sodium dodecyl sulfate and subsequent digestion of the sodium dodecyl sulfate-insoluble residue with proteases and alpha-amylase. Cell wall fractions obtained by sodium dodecyl sulfate extraction and protease digestion were highly contaminated by a glucose polymer. The polyglucose was removed by alpha-amylase treatment, and the peptidoglycans were left behind. Analyses with amino acids and amino sugars of the cell wall fractions and peptidoglycan specimens revealed that D-glutamic acid, D-alanine, L-alanine, meso-2,6-diaminopimelic acid (A2pm), muramic acid, and glucosamine were the principal components. The dinitrophenylation method revealed that about half of the A2pm residue had a free amino group, and analysis by hydrazinolysis showed that a small part of alanine and A2pm was located at the C terminal. The above results indicate that one of the amino groups of the A2pm residue at one strand of the stem peptide subunit cross-linked to the carboxyl group of alanine of the neighboring strand. It was thus revealed that the peptidoglycans of L. buccalis belong to the A1 gamma type of the classification by Schleifer and Kandler (Bacteriol. Rev. 36:407-477).