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 ×
108 cells/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.