Fig. 2

B. burgdorferi B31 BB0838 is surface localized and amphiphilic. a. bb0838 is in an operon with uvrA and uvrB. Schematic of the uvrB, uvrA, and bb0838 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 uvrB and uvrA (primers 1 and 2, left panel) and uvrA and bb0838 (primers 3 and 4, right panel). A negative control lacking 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 was performed to separate soluble, aqueous (A) phase proteins from amphiphilic, detergent (D) phase proteins. Aqueous and detergent fractions were separated by SDS-PAGE and immunoblotted with anti-BB0838 peptide antibodies. Equivalent fractions were also immunoblotted with anti-BamB and anti-Skp antibodies as detergent-enriched and aqueous-enriched controls, respectively. c-d. Whole-cell lysates were washed and incubated with (c) proteinase K (PK) or (d) trypsin. Samples were then immunoblotted with BB0838 peptide antibodies to assess surface degradation of BB0838. Equivalent samples were also immunoblotted with OspA antibodies or P66 antibodies for PK and trypsin experiments, respectively, 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. Molecular weight standards (in kDa) are shown at left