Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli.
Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.
Li
Shi-Yan
SY
Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, ND 58202-9037, USA.
Höltje
Joachim-Volker
JV
Young
Kevin D
KD
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics; D015394:Molecular Structure; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000032:analysis* / Q000737:chemistry; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C478706:penicillin-binding protein 4, E coli; D002267:Muramoylpentapeptide Carboxypeptidase