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Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin
Universität Würzburg, Germany.
Universität Würzburg and Jacobs University Bremen, Germany.
Universität Würzburg, Germany.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The outer membrane protein P66 of the Lyme disease spirochete Borrelia burgdorferi is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl. We studied in a non-theoretical manner the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. Furthermore, we calculated the filling of the channel with these nonelectrolytes and the results revealed that nonelectrolytes with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. Thus, the diameter of the P66 entrance was determined to be ≤ 1.9 nm with a constriction site diameter of about 0.7 nm. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% after addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose in the low millimolar range. Interestingly, the analysis of the power density spectra of P66 after blockage with nonelectrolytes revealed no chemical interaction responsible for channel block. The blockage of one P66 single-channel conductance unit of 11 nS occurred by seven subconducting states, thus indicating a heptameric organization of the P66 oligomer. This organization of P66 as a heptamer was confirmed by Blue Native PAGE and immunoblot analysis, which demonstrated that P66 forms a complex with a mass of approximately 460 kDa.

Keyword [en]
nutrient uptake, outer membrane transport, Lyme disease, porin radius determination, black lipid bilayer
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
URN: urn:nbn:se:umu:diva-32783OAI: diva2:305738

Ignas Bunikis är ej medförfattare till den publicerade versionen i PLoS ONE 2013, 8:11, Article Number: e78272, DOI: 10.1371/journal.pone.0078272

Ignas Bunikis is not co-author in the published version in PLoS ONE 2013, 8:11, Article Number: e78272, DOI: 10.1371/journal.pone.0078272

Available from: 2010-03-25 Created: 2010-03-25 Last updated: 2013-12-11Bibliographically approved
In thesis
1. Borrelia channel-forming proteins: structure and function
Open this publication in new window or tab >>Borrelia channel-forming proteins: structure and function
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B. crocidurae. Both diseases differ in their manifestations, duration times and dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known.

Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species.

The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B. duttonii, B. recurrentis and B. hermsii were compared and their biophysical properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2010. 77 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1340
Borrelia, Lume disease, relapsing fever, BesC, drug efflux, DipA, P66, porins
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-32777 (URN)978-91-7264-971-2 (ISBN)
Public defence
2010-04-16, Betula Lecture Hall, Umeå University, Umeå, 10:00 (English)
Available from: 2010-03-26 Created: 2010-03-25 Last updated: 2010-03-26Bibliographically approved

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