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Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

ABSTRACT: The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

SUBMITTER: Skare JT

PROVIDER: S-EPMC178274 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

Skare J T JT Champion C I CI Mirzabekov T A TA Shang E S ES Blanco D R DR Erdjument-Bromage H H Tempst P P Kagan B L BL Miller J N JN Lovett M A MA

Journal of bacteriology 19960801 16

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we f ...[more]

PMID: 8759855

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Project description:Among bacterial cell envelopes, the Borrelia burgdorferi outer membrane (OM) is structurally unique in that the identities of many protein complexes remain unknown; however, their characterization is the first step toward our understanding of membrane protein interactions and potential functions. Here, we used two-dimensional blue native/SDS-PAGE/mass spectrometric analysis for a global characterization of protein-protein interactions as well as to identify protein complexes in OM vesicles isolated from multiple infectious sensu stricto isolates of B. burgdorferi. Although we uncovered the existence of at least 10 distinct OM complexes harboring several unique subunits, the complexome is dominated by the frequent occurrence of a limited diversity of membrane proteins, most notably P13, outer surface protein (Osp) A, -B, -C, and -D and Lp6.6. The occurrence of these complexes and specificity of subunit interaction were further supported by independent two-dimensional immunoblotting and coimmunoprecipitation assays as well as by mutagenesis studies, where targeted depletion of a subunit member (P66) selectively abolished a specific complex. Although a comparable profile of the OM complexome was detected in two major infectious isolates, such as B31 and 297, certain complexes are likely to occur in an isolate-specific manner. Further assessment of protein complexes in multiple Osp-deficient isolates showed loss of several protein complexes but revealed the existence of additional complex/subunits that are undetectable in wild-type cells. Together, these observations uncovered borrelial antigens involved in membrane protein interactions. The study also suggests that the assembly process of OM complexes is specific and that the core or stabilizing subunits vary between complexes. Further characterization of these protein complexes including elucidation of their biological significance may shed new light on the mechanism of pathogen persistence and the development of preventative measures against the infection.

Dataset Information

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

Publications

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

Similar Datasets

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