Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi

Archie Digital Collections

Show simple item record Kumru, Ozan S Schulze, Ryan J Slusser, Joyce G Zuckert, Wolfram R 2011-01-07T17:06:24Z 2011-01-07T17:06:24Z 2010-11-03
dc.description.abstract Abstract Background In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins. Results A Glu-Asp codon pair in the inner membrane-localized OspA20:mRFP1 fusion was chosen for mutagenesis since the two negative charges were previously shown to define the phenotype. A library of random mutants in the two codons was generated and expressed in B. burgdorferi. In situ surface proteolysis combined with fluorescence activated cell sorting (FACS) was then used to screen for viable spirochetes expressing alternative subsurface OspA:mRFP1 fusions. Analysis of 93 clones randomly picked from a sorted cell population identified a total of 43 distinct mutants. Protein localization assays indicated a significant enrichment in the selected subsurface phenotype. Interestingly, a majority of the subsurface mutant proteins localized to the outer membrane, indicating their impairment in "flipping" through the outer membrane to the spirochetal surface. OspA20:mRFP1 remained the protein most restricted to the inner membrane. Conclusions Together, these results validate this FACS-based screen for lipoprotein localization and suggest a rather specific inner membrane retention mechanism involving membrane anchor-proximal negative charge patches in this model B. burgdorferi lipoprotein system.
dc.title Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi
dc.type Journal Article 2010-11-17T07:01:31Z
dc.description.version Peer Reviewed
dc.language.rfc3066 en
dc.rights.holder Kumru et al.; licensee BioMed Central Ltd.

Files in this item

This item appears in the following Collection(s)

Show simple item record

Search Archie

Advanced Search


My Account

Sword Copy