The Unique N-Terminal Domain of Chlamydial Bactofilin Mediates Its Membrane Localization and Ring-Forming Properties

Chlamydia trachomatis is an obligate intracellular bacterial pathogen. In evolving to the intracellular niche, Chlamydia has reduced its genome size compared to other bacteria and, as a consequence, has a number of unique features. For example, Chlamydia engages the actin-like protein MreB, rather than the tubulin-like protein FtsZ, to direct peptidoglycan (PG) synthesis exclusively at the septum of cells undergoing polarized cell division. Interestingly, Chlamydia possesses another cytoskeletal element-a bactofilin ortholog, BacA. Recently, we reported BacA is a cell size-determining protein that forms dynamic membrane-associated ring structures in Chlamydia that have not been observed in other bacteria with bactofilins. Chlamydial BacA possesses a unique N-terminal domain, and we hypothesized this domain imparts the membrane-binding and ring-forming properties of BacA. We show that different truncations of the N terminus result in distinct phenotypes: removal of the first 50 amino acids (Delta N50) results in large ring structures at the membrane whereas removal of the first 81 amino acids (Delta N81) results in an inability to form filaments and rings and a loss of membrane association. Overexpression of the Delta N50 isoform altered cell size, similar to loss of BacA, suggesting that the dynamic properties of BacA are essential for the regulation of cell size. We further show that the region from amino acid 51 to 81 imparts membrane association as appending it to green fluorescent protein (GFP) resulted in the relocalization of GFP from the cytosol to the membrane. Overall, our findings suggest two important functions for the unique N-terminal domain of BacA and help explain its role as a cell size determinant.IMPORTANCE Bacteria use a variety of filament-forming cytoskeletal proteins to regulate and control various aspects of their physiology. For example, the tubulin-like FtsZ recruits division proteins to the septum whereas the actin-like MreB recruits peptidoglycan (PG) synthases to generate the cell wall in rod-shaped bacteria. Recently, a third class of cytoskeletal protein has been identified in bacteria-bactofilins. These proteins have been primarily linked to spatially localized PG synthesis. Interestingly, Chlamydia, an obligate intracellular bacterium, does not have PG in its cell wall and yet possesses a bactofilin ortholog. In this study, we characterize a unique N-terminal domain of chlamydial bactofilin and show that this domain controls two important functions that affect cell size: its ring-forming and membrane-associating properties. Bacteria use a variety of filament-forming cytoskeletal proteins to regulate and control various aspects of their physiology. For example, the tubulin-like FtsZ recruits division proteins to the septum whereas the actin-like MreB recruits peptidoglycan (PG) synthases to generate the cell wall in rod-shaped bacteria.