Purification and structure elucidation of the N-acetylbacillosamine-containing polysaccharide from Bacillus licheniformis ATCC 9945.

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4-acetamido-2-amino-2,4,6-trideoxy-D-glucose, termed N-acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2-acetamido-4-amino-2,4,6-trideoxy-D-glucose, was found in a surface layer (S-layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S-layer lattice, indicating that the N-acetylbacillosamine present in that organism might be a constituent of a cell wall-associated polymer. For elucidation of the structure of the N-acetylbacillosamine-containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S-layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4-diamino-2,4,6-trideoxy-glucose, 2-acetamido-2-deoxy-glucose, 2-acetamido-2-deoxy-galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One- and two-dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: [-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-alpha-D-GlcpNAc-(1-->3)-beta-D-Bacp2N4NAc-(1-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-beta-D-GalpNAc-(1-->](n)