Localization of core lipopolysaccharide on the inner membrane of Salmonella typhimurium
The mechanism of lipopolysaccharide (LPS) translocation to the outer membrane (OM) is poorly defined. The authors have used affinity purified antiR/sub a/ LPS-IgG in immunoelectron microscopy and /sup 125/I protein A radio-labeling experiments to localize newly synthesized R/sub a/ LPS on the inner and outer faces of the inner membrane (IM)/sup a/. Cells were pulsed with galactose for 5 min to permit synthesis of R/sub a/ LPS and chased 0-20' by dilution into glucose medium. LPS translocation was halted by addition of DNP at 0/sup 0/. Samples +/-gal were spheroplasted or french pressed to expose outer and inner faces of the IM respectively. Immunoelectron microscopy employed ferritin conjugated goat anti-rabbit IgG as secondary Ab. Galactose pulsed cells showed no apparent labeling of core-LPS on the outer face of the IM. The outer face of the OM was well decorated. French pressed IM vesicles were purified by isopycnic sucrose gradient centrifugation and incubated sequentially with anti-R/sub a/ LPS and /sup 125/I protein A. /sup 125/I labeling was increased 5-fold over gal-negative controls, consistent with exposure of R/sub a/ LPS on the inner face of the IM. IM isolated after treatment of intact spheroplasts with anti-R/sub a/ LPS and /sup 125/I protein A showed no /sup 125/I labeling, although good radiolabeling of OM was found in all cases. These results suggest that core LPS is synthesized on the inner face of the IM and does not accumulate on the outer face in amounts detectable by these methods before translocation to the OM.
- Research Organization:
- Univ. of Connecticut Health Center, Farmington
- OSTI ID:
- 5334166
- Report Number(s):
- CONF-8606151-; TRN: 86-031418
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL MEMBRANES
CHEMICAL COMPOSITION
LIPOPOLYSACCHARIDES
BIOLOGICAL LOCALIZATION
MEMBRANE TRANSPORT
AFFINITY
CENTRIFUGATION
ELECTRON MICROSCOPY
GALACTOSE
GOATS
IMMUNOGLOBULINS
IODINE 125
PROTEINS
RABBITS
SALMONELLA TYPHIMURIUM
TRACER TECHNIQUES
TRANSLOCATION
ALDEHYDES
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
CARBOHYDRATES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
GLOBULINS
HEXOSES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIPIDS
MAMMALS
MEMBRANES
MICROORGANISMS
MICROSCOPY
MONOSACCHARIDES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
POLYSACCHARIDES
RADIOISOTOPES
RUMINANTS
SACCHARIDES
SALMONELLA
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques
550701 - Microbiology- Tracer Techniques