A SIMPLE GEL-ELECTROPHORETIC METHOD FOR ANALYZING THE MUROPEPTIDE COMPOSITION OF BACTERIAL PEPTIDOGLYCAN

The muropeptide composition of bacterial peptidoglycan is currently mo st efficiently determined by reverse-phase high-pressure liquid chroma tography (HPLC). Though sensitive, the HPLC procedure is technically d emanding and has been applied to a relatively small number of bacteria l strains and species, We have found that fluorescence-assisted carboh ydrate electrophoresis (FACE) is a simple, rapid method hy which reduc ing muropeptides from multiple peptidoglycan samples can be,visualized . Individual reducing muropeptides were covalently labeled with the fl uorescent molecule 8-aminonaphthalene-1,3,6-trisulfonic acid, after wh ich they were separated by electrophoresis through a 35% polyacrylamid e gel and visualized by exposure to UV light. FACE detected the approp riate numbers of reducing muropeptides in the proper proportions for f our bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterobacter c loacae, and Yersinia enterocolitica, Bs little as 2 to 5 pmol per muro peptide was detected when the intensity of the fluorescent signal was measured dth a charge-coupled device camera, at a level of sensitivity between 50 and 250 times higher than that of the classic HPLC techniq ue, Thus, FACE may be used to identify interesting peptidoglycan sampl es prior Lo more-extensive analysis by HPLC, or FACE may eventually re place HPLC for some applications.