Abstract
Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.
Highlights
Bactofilins are a newly identified class of proteins sharing the bactofilin (DUF583) domain and are widespread in most bacterial species analyzed so far [1, 2]
Based on the presence of a DUF583 domain, two genes, yhbE and yhbF, can be identified to encode the bactofilins BacE and BacF, respectively, in Bacillus subtilis. Both genes are in a putative operon together with the upstream yhbD open reading frame, which is only conserved in Bacilli and in the closely related Clostridia [29], whereas bactofilins genes are found in many bacteria
In the reciprocal experiments (Fig 7C, lower panels), BacE did not bind to the column, but eluted together with 6-His-BacF. These results show that BacE and BacF interact with each other in vitro, and are able to form filamentous structures when expressed at high levels, there is no evidence for filament formation in B. subtilis cells at physiological expression levels (Fig 5A–5C)
Summary
Bactofilins are a newly identified class of proteins sharing the bactofilin (DUF583) domain and are widespread in most bacterial species analyzed so far [1, 2]. Many of the genomes containing bactofilin genes have multiple paralogous copies, which can be organized in an operon-like structure, or can be present at distinct genomic sites. In Caulobacter crescentus and in Myxococcus xanthus, bactofilins recruit other proteins to specific subcellular sites: a cell wall synthetic.
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