PlzA binds B. burgdorferi RNAs in vitro
To determine if PlzA can bind B. burgdorferi RNA, we performed anin vitro RNA-binding assay using PlzA as bait. His-tagged wild-type and mutant PlzA proteins were immobilized on a cobalt column and B. burgdorferi cleared lysates were passed over the column (Fig. 6A). Columns were washed and PlzA was eluted with imidazole. Eluates were immunoblotted to assay for PlzA protein (Fig. 6B), and RNA bound to PlzA was isolated and run on a bioanalyzer (Fig. 6C). Immunoblot analyses demonstrate that PlzA proteins were found in both eluates but were more concentrated in the first fraction (Fig. 6B, E1). We found that wild-type PlzA and PlzA R145D, which does not bind c-di-GMP, both bind B. burgdorferi RNA, while neither of the PlzA triple mutant proteins nor the control without PlzA protein bind RNA (Fig. 6C). These results support the hypothesis that the aromatic amino acids in the N - and C -terminal domains are important for binding RNA and corroborate the in vitro filter binding data. These results also demonstrate that the positive surface charges of PlzA are not sufficient to bind B. burgdorferi RNA. Notably, the RNA detected on the bioanalyzer was relatively small (25-150 nucleotides), which suggest PlzA binds predominantly sRNAs, but the specific RNAs bound by PlzA awaits further investigation.