Article Figures & Data
Figures
- FIG. 1.
Double-labeling IFAs of B. burgdorferi. (A) Specificity of MAb B11 for ErpA/I/N and of each polyclonal antibody raised against ErpB/J/O, ErpG, ErpK, ErpL, ErpM, ErpP, ErpX, and ErpY. Bacteria were cultured at 34°C, and lysates were analyzed by immunoblot with each antibody preparation. Number of kilodaltons is given on left. (B) IFA of fixed B. burgdorferi labeled with MAb B11 (left column) and labeled with a polyclonal antibody against a specific Erp protein (middle column). Both IFAs were analyzed (right column).
- FIG. 2.
Effect of culture temperature on supercoiled circular DNA. Plasmid DNAs from bacteria grown at 23 or 34°C were separated on agarose gels containing either 0, 3, 9, or 12 μg of chloroquine/ml, followed by Southern blot analysis. Each experiment was repeated twice, and arrows indicate supercoiled (SC) DNA. In some panels, linearized plasmid is also visible. Top row, membranes probed with probe derived from ORF3-ORFC region encoded on cp32-1 (15), which is specific for that plasmid. Middle row, membranes probed with the generic cp32 probe 2, which recognizes every erp promoter region (26). Lowest row, membranes probed with probe 3, derived from the ospC promoter region of cp26 (6).
- FIG. 3.
Effects of coumermycin A1 on protein expression. B. burgdorferi was incubated with 0.001, 0.003, 0.01, 0.03, and 0.1 μg of coumermycin A1/ml for 30 min, 4 h, and 4 days, followed by Western blot analysis. Top row, membranes incubated with MAb B11 directed against ErpA/I/N. Lower row, membranes incubated with MAb B5 directed against OspC.
- FIG. 4.
Orientation of the erp, bppA, bppB, and bppC loci on B. burgdorferi cp32 (drawn to scale). erp loci may be either mono- or bicistronic. The start codon of bppC is located approximately 430 bp from the start codon of its adjacent erp locus. bppA encodes a 51-kDa protein, bppB encodes a 17-kDa protein, and bppC encodes a 32-kDa protein.
- FIG. 5.
Western blot analysis of ErpA/I/N, BppA, BppB, and BppC expression by B. burgdorferi. Left column, bacteria cultured at 23°C or shifted from 23 to 35°C. Middle column, bacteria cultured at pH 7.0 or 8.0. Right column, bacteria treated with MNNG (+) or left untreated (−). nd, not done.
- FIG. 6.
Triton X-114 extraction of B. burgdorferi cells. Fractions were separated into three different phases. The detergent phase contains the outer membrane proteins. The aqueous phase contains periplasmic proteins. The pellet contains the protoplasmic cylinder (cytoplasm, the inner membrane, and the anchored periplasmic flagella).
Tables
- TABLE 1.
Oligonucleotides used in these studies
Primer Sequence (5′ to 3′) Use for PCR ORFD-1 ACGATAGGGTAATATCAAAAAGG cp32-1 ORFD-2 AGTTCATCTAATAAAAATCCCGTG cp32-1 E-506 AACTTTTTTTTACATCTTCACCAC B31 erpK promoter E-513 CTGTTTGTTAATATGTAATAGCTG B31 erpK promoter PC-29 TCTCTAATTCTTCTTGCAATTAGTTG ospC promoter PC-40 TCCTGAATTATTACAAGATATAAATA ospC promoter
