Abstract
Bactofilins are a recently discovered class of cytoskeletal protein, widely implicated in subcellular organization and morphogenesis in bacteria and archaea. Several lines of evidence suggest that bactofilins polymerize into filaments using a central β-helical core domain, flanked by variable N- and C-terminal domains that may be important for scaffolding and other functions. However, a systematic exploration of the characteristics of these domains has yet to be performed. In Asticcacaulis biprosthecum, the bactofilin BacA serves as a topological organizer of stalk synthesis, localizing to the stalk base and coordinating the synthesis of these long, thin extensions of the cell envelope. The easily distinguishable phenotypes of wild-type A. biprosthecum stalks and ΔbacA "pseudostalks" make this an ideal system for investigating how mutations in BacA affect its functions in morphogenesis. Here, we redefine the core domain of A. biprosthecum BacA using various bioinformatics and biochemical approaches to precisely delimit the N- and C- terminal domains. We then show that loss of these terminal domains leads to cells with severe morphological abnormalities, typically presenting a pseudostalk phenotype. BacA mutants lacking the N- and C- terminal domains also exhibit localization defects, implying that the terminal domains of BacA may be involved in its subcellular positioning, whether through membrane interactions through the N-terminal domain or through interactions with the stalk-specific morphological regulator SpmX through the C-terminal domain. We further show that point mutations that render BacA defective for polymerization lead to stalk synthesis defects. Overall, our study suggests that BacA's polymerization, membrane association, and interactions with other morphological factors all play a crucial role in the protein's function as a morphogenic regulator. The specialization and modularity of the terminal domains may underlie the remarkable functional versatility of the bactofilins in different species.