Bactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacA, in the obligate intracellular pathogen species are obligate intracellular bacteria that undergo a developmental cycle alternating between infectious, non-dividing EBs (elementary bodies) and non-infectious, dividing RBs (reticulate bodies). As divides by a polarized division process, we hypothesized that BacA may function to establish polar... More
Bactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacA, in the obligate intracellular pathogen species are obligate intracellular bacteria that undergo a developmental cycle alternating between infectious, non-dividing EBs (elementary bodies) and non-infectious, dividing RBs (reticulate bodies). As divides by a polarized division process, we hypothesized that BacA may function to establish polarity in these unique bacteria. Utilizing a combination of fusion constructs and high-resolution fluorescence microscopy, we determined that BacA forms dynamic, membrane-associated filament- and ring-like structures in replicative RB form. Contrary to our hypothesis, these structures are distinct from the microbe's cell division machinery and do not colocalize with septal peptidoglycan or MreB, the major organizer of the bacterium's division complex. Bacterial two-hybrid assays demonstrated BacA interacts homotypically but does not directly interact with proteins involved in cell division or peptidoglycan biosynthesis. To investigate the function of BacA in chlamydial development, we constructed a conditional knockdown strain using a newly developed CRISPR interference system. We observed that reducing expression significantly increased chlamydial cell size. Normal RB morphology was restored when an additional copy of was expressed during knockdown. These data reveal a novel function for chlamydial bactofilin in maintaining cell size in this obligate intracellular bacterium.
