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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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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<jats:italic>B. burgdorferi<\/jats:italic>.<jats:italic>In situ<\/jats:italic>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>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<jats:italic>B. burgdorferi<\/jats:italic>lipoprotein system.<\/jats:p><\/jats:sec>","DOI":"10.1186\/1471-2180-10-277","type":"journal-article","created":{"date-parts":[[2010,11,3]],"date-time":"2010-11-03T19:30:14Z","timestamp":1288812614000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi"],"prefix":"10.1186","volume":"10","author":[{"given":"Ozan S","family":"Kumru","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryan J","family":"Schulze","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joyce G","family":"Slusser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wolfram R","family":"Z\u00fcckert","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2010,11,3]]},"reference":[{"key":"1233_CR1","doi-asserted-by":"publisher","first-page":"561","DOI":"10.1172\/JCI9427","volume":"106","author":"U Pal","year":"2000","unstructured":"Pal U, de Silva AM, Montgomery RR, Fish D, Anguita J, Anderson JF, Lobet Y, Fikrig E: Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A. 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