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Research Article
1 October 1994

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii

Abstract

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.

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Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 176Number 19October 1994
Pages: 6088 - 6099
PubMed: 7928971

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Published online: 1 October 1994

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Authors

D R Blanco
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
K Reimann
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
J Skare
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
C I Champion
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
D Foley
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
M M Exner
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
R E Hancock
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
J N Miller
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
M A Lovett
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.

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