A simple gel electrophoretic method for analyzing the muropeptide composition of bacterial peptidoglycan.
The muropeptide composition of bacterial peptidoglycan is currently most efficiently determined by reverse-phase high-pressure liquid chromatography (HPLC). Though sensitive, the HPLC procedure is technically demanding and has been applied to a relatively small number of bacterial strains and species. We have found that fluorescence-assisted carbohydrate electrophoresis (FACE) is a simple, rapid method by which reducing muropeptides from multiple peptidoglycan samples can be visualized. Individual reducing muropeptides were covalently labeled with the fluorescent molecule 8-aminonaphthalene-1,3,6-trisulfonic acid, after which they were separated by electrophoresis through a 35% polyacrylamide gel and visualized by exposure to UV light. FACE detected the appropriate numbers of reducing muropeptides in the proper proportions for four bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Yersinia enterocolitica. As little as 2 to 5 pmol per muropeptide was detected when the intensity of the fluorescent signal was measured with a charge-coupled device camera, at a level of sensitivity between 50 and 250 times higher than that of the classic HPLC technique. Thus, FACE may be used to identify interesting peptidoglycan samples prior to more-extensive analysis by HPLC, or FACE may eventually replace HPLC for some applications.
Young
K D
KD
Department of Microbiology and Immunology, School of Medicine, University of North Dakota, Grand Forks, North Dakota 58202-9037, USA. kyoung@mail.med.und.nodak.edu
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000032:analysis; D004586:Electrophoresis / Q000379:methods*; D016972:Enterobacter cloacae / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000494:pharmacology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D010457:Peptidoglycan / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012680:Sensitivity and Specificity; D015008:Yersinia enterocolitica / Q000737:chemistry*
D004187:Disaccharides; D005456:Fluorescent Dyes; D009281:Naphthalenes; D010457:Peptidoglycan; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D009113:Muramidase