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The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13.
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
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2006 (English)In: Journal of Bacteriology, ISSN 0021-9193, Vol. 188, no 12, 4207-4217 p.Article in journal (Refereed) Published
Abstract [en]

The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13. Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.

Place, publisher, year, edition, pages
2006. Vol. 188, no 12, 4207-4217 p.
Keyword [en]
Bacterial Outer Membrane Proteins/*metabolism, Borrelia burgdorferi/*metabolism/physiology, Carboxypeptidases/metabolism, Ion Channels/*metabolism, Multigene Family, Peptide Hydrolases/metabolism
URN: urn:nbn:se:umu:diva-12925DOI: doi:10.1128/JB.00302-06PubMedID: 16740927OAI: diva2:152596
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2009-10-28Bibliographically 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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