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Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
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2002 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, 6811-6819 p.Article in journal (Refereed) Published
Abstract [en]

P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.

Place, publisher, year, edition, pages
2002. Vol. 184, no 24, 6811-6819 p.
National Category
Cell and Molecular Biology
URN: urn:nbn:se:umu:diva-5029DOI: 10.1128/JB.184.24.6811-6819.2002PubMedID: 12446631OAI: diva2:144378
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2011-10-26Bibliographically approved
In thesis
1. Porins of Borrelia burgdorferi
Open this publication in new window or tab >>Porins of Borrelia burgdorferi
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Borrelia burgdorferi is a pathogenic spirochete which cycles between its arthropod vector and vertebrate host. If transmitted to humans, B. burgdorferi causes Lyme disease, an infection which can impair different organs, such as the skin, joints, nervous system and heart. Alterations in protein expression due to the different environments Borrelia encounters during its complicated life cycle require advanced adaptation mechanisms. The outer surface-exposed proteins play a critical role in survival and pathogenesis of Borrelia in different hosts and tissues, being involved in avoiding the host immune response, adhesion to different tissues and nutrient acquisition. This thesis aimed to characterize integral outer membrane proteins which play a role in solute and nutrient uptake, and provides support for their role in the environmental adaptation of Borrelia.

In this thesis, three B. burgdorferi proteins, P13, BBA01 and P66, were shown to be porins, and characterized structurally and functionally using a combination of biochemical, biophysical and genetic methods. The channel-forming function of the 13 kDa protein, P13, was elucidated by a lipid bilayer assay. Post-translational processing of P13 occurred at the C-terminus by C-terminal processing protease (CtpA)-dependent cleavage. The membrane-spanning architecture of P13 was determined by epitope mapping and computer-based structural predictions which revealed that P13 is an unusual porin, not possessing the structural properties of conventional porins: rather than forming β-barrels, it is predicted to span the membrane with hydrophobic α-helices.

p13 belongs to a paralogous gene family. The transcription of p13 and other gene family members during in vitro growth and in a mouse infection model was therefore investigated. The paralog BBA01, which has the highest sequence homology to P13, is expressed during in vitro growth in all three Lyme disease causing species, although at very low levels. Like P13, BBA01 is also processed by CtpA and exhibits very similar channel-forming activity. Furthermore, in the absence of P13, a proportion of total BBA01 protein is relocated to the bacterial surface with strong indications that BBA01 and P13 are functionally interchangeable.

P66, an integrin binding protein, was also determined to be a porin. The oligomeric state of native P66, elucidated by chemical cross-linking, indicated that P66 forms trimers, as do the majority of conventional porins. Electron crystallography and a projection map of P66 crystals at 2.2 nm resolution revealed tetragonal unit cell symmetry with the area intercalated between the assembled protein structures consistent with the approximate expected size of the channel formed by P66. Finally, the biological relevance of two porins, P13 and P66, was demonstrated in a double mutant displaying a stress response as revealed by increased sensitivity to high osmolarity and elevated expression of the B. burgdorferi heat-shock protein HtrA homolog.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi, 2006. 84 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1019
Lyme disease, P13, paralog, P66, channel-forming activity, porin
National Category
Microbiology in the medical area
urn:nbn:se:umu:diva-740 (URN)91-7264-059-6 (ISBN)
Public defence
2006-04-21, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2006-03-31 Created: 2006-03-31 Last updated: 2009-10-28Bibliographically approved

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