PlzA RNA binding assay
The Pierce His protein interaction pulldown kit was used following the manufacturer’s instructions with some modifications. Briefly, spin columns were prepared with 50 µl of slurry and equilibrated with a 1:1 TBS: Pierce Lysis Buffer with 10 mM imidazole. 150 µg of recombinant His-tagged PlzA proteins were immobilized on the slurry by incubating in the spin columns for 30 min at 4°C on a rotating platform. Unbound protein was removed and columns were washed. 50 ml of B31 5A4 B. burgdorferi were grown to a cell density of 1 × 10cells/ml in BSK-II at 34°C and 5% CO2. Cells were pelleted at 5,000 × g and frozen at -80°C. Pellets were thawed on ice and resuspended in wash solution (1:1 ratio of Pierce lysis buffer and BupH Tris Buffered Saline; 500 µl total volume). Cells were lysed in a 2-ml microfuge tube with 500 µl of 0.1 mm glass beads with a Retsch at 30 Hz for 5-10 min in a frozen cassette. Lysates were cleared at 12000 × g for 10 min, added to the spin column and rotated for 1 h at 4°C. Columns were centrifuged at 1250 × g for 30 sec and flow throughs were saved for SDS-PAGE analysis. The columns were washed five times with 400 µl of wash solution and centrifuged at 1250 × g for 30 s. Proteins were eluted with 250 µl of wash buffer containing 300 mM imidazole. The columns were rotated at 4°C for 5 min and then centrifuged at 1250 ×g for 30 s. Two eluates were acquired per sample. 10 µl of each eluate was saved for SDS-PAGE analysis. The remaining sample was prepared for hot phenol RNA isolation as previously described in Lybecker et al. (75). RNA concentration was measured using a Nanodrop spectrophotometer and analyzed on an Agilent 2100 Bioanalyzer picochip.