Fig. 3

B. burgdorferi B31 BB0406 is amphiphilic and OM-associated. a. bb0404, bb0405, and bb0406 are transcribed as an operon. Schematic of the bb0404, bb0405, and bb0406 operon is shown in the top panel. Total RNA was isolated from B. burgdorferi B31 cells and used for RT-PCR using primer pairs listed in Table 3. Primer pairs were used that amplify a region traversing bb0404 and bb0405 (primers 5 and 6, left panel) and bb0405 and bb0406 (primers 7 and 8, right panel). A negative control without RT was used as template for the RT-PCR (−RT) as was as a positive control in which genomic DNA instead of cDNA was used as template (DNA). b. Triton X-114 phase partitioning of B. burgdorferi B31 whole-cell lysates were performed to separate aqueous-enriched (A) proteins from detergent-enriched (D) proteins. Aqueous and detergent fractions were separated by SDS-PAGE and immunoblotted with rat anti-BB0405 and rat anti-BB0406 antibodies. As controls, equivalent fractions were also immunoblotted with antibodies directed against the detergent-soluble lipoprotein BamB and the soluble, periplasmic protein Skp. c. Outer membrane (OM) and protoplasmic cylinder (PC) fractions were isolated from B. burgdorferi B31. Subsequently, OM and PC fractions were immunoblotted with rat anti-BB0405 and anti-BB0406 antibodies. Equivalent membranes were also subjected to immunoblot with BamA and OppAIV antibodies. d. Whole-cell lysates were washed and incubated with proteinase K (PK). Samples were then immunoblotted with BB0405 or BB0406 antibodies to assess surface degradation of the protein. Equivalent samples were also immunoblotted with OspA antibodies to control for protease activity and with antibodies that recognize the periplasmic protein FlaB to ensure that the OM remained intact throughout the proteolysis experiments