Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis.
Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain (B. fragilis CFPL 358) and two susceptible strains of B. fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60-67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60-67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.
Laboratoire de Bactériologie, Faculté des Sciences Pharmaceutiques et Biologiques, Lille, France.
M F
MF
Odou
E
E
Singer
M B
MB
Romond
L
L
Dubreuil
D001441:Bacteroides fragilis / Q000737:chemistry* / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D011510:Proteolipids / Q000502:physiology
D018272:Porins; D011510:Proteolipids; C020485:proteoliposomes