Characterization of the DUF4004 protein YhbD of Bacillus subtilis: A connection to bactofilins and vancomycin-mediated switch of YhbD between membrane localization and DNA binding

YhbD from Bacillus subtilis is a member of the poorly characterized DUF4004 protein family, which is conserved in Firmicutes. So far, only the protein Cap0037 from Clostridium acetobutylicum has been described as an autoregulatory and global transcription factor. YhbD is expressed in the operon with the two bactofilin-coding genes bacE and bacF. Bactofilins are involved in central cellular processes in many bacteria, such as motility and cell wall synthesis, by acting as scaffold proteins that recruit enzymes and regulators to defined locations, contributing to the structural organization of the cell. In B. subtilis, BacE and BacF are essential for the formation of the hook and filament structures of the flagellum. In double mutants, these structures are entirely absent although the basal bodies remain intact. If there is a functional link between YhbD and the bactofilins BacE and BacF, it remains unclear. This work provides new functional insights into the DUF4004 protein family, as YhbD from B. subtilis is the second experimentally investigated member of this family. Like Cap0037, a helix-turn-helix motif was identified in YhbD. In vitro, I demonstrated DNA binding for the homologous protein GtYhbD from Geobacillus thermodenitrificans, including binding to the promoter region of the yhbD-bacEF operon. However, a specific DNA binding site has not yet been determined. Despite the genomic coupling of yhbD with the bactofilin-coding genes bacE and bacF, no direct effect of YhbD on swimming motility could be observed in this work. However, I was able to demonstrate a correlation between the subcellular localization of YhbD and the expression of the bactofilins, as well as the genomic positioning of the genes. Under conditions that promote bactofilin expression, YhbD localized to static, membrane-associated foci resembling BacF. By repressed expression of bacE and bacF, or ectopic expression of yhbD at another genomic position, the localization of YhbD shifted to a diffuse distribution in the nucleoid region. This observation suggests that the genomic proximity of yhbD to bacE and bacF is functionally relevant, and the expression of these adjacent genes significantly influences the localization of YhbD. Moreover, I was able to demonstrate a direct interaction of GtYhbD with GtBacE and GtBacF in vitro. This binding led to a significant reduction in the DNA binding activity of GtYhbD, likely due to steric hindrance. The DNA binding ability of YhbD was also confirmed, with YhbD binding to the promoter region of the yhbD-bacEF operon. However, it remains unclear whether YhbD remains exclusively fixed to the membrane through its interaction with bactofilins or whether it also associates directly with the membrane, for example, through lipophilic interactions. In addition to bactofilin-dependent localization, I observed a change in the distribution of YhbD under vancomycin-induced cell wall stress: the previously membrane-associated foci dissolved, giving way to a diffuse localization in the nucleoid. This observation could indicate a possible switch between YhbD's bactofilin-bound, presumably inactive form and its active, DNA-binding form. For other bactofilin-associated systems, an influence on cell wall synthesis has already been described, supporting the hypothesis that YhbD, together with bactofilins, may be involved in regulating or recruiting enzymes for cell wall biosynthesis, particularly under conditions of cell wall stress. A possible connection to regulatory networks of flagellar assembly is suggested by previous studies, which described an interaction between BacE and the flagellar regulator FliW. FliW controls the post-transcriptional regulation of flagellin synthesis by binding to the translation repressor CsrA. It has also been demonstrated that FliW displaces BacE at high concentrations of flagellin. This leads to the hypothetical model that, under conditions of disrupted flagellar assembly, such as those caused by cell wall stress, Flagellin-FliW complexes accumulate and displace BacE from its interaction with FliW. In this context, YhbD could be released, allowing bactofilins to recruit enzymes for cell wall biosynthesis to the membrane, while YhbD binds to DNA in its active form. Whether YhbD detects the signal for disrupted cell wall biosynthesis directly, via its membrane association, or indirectly through bactofilins, remains unclear. The results of this study suggest that YhbD, as the second characterized member of the DUF4004 protein family, acts as a sensor-regulator protein, forming an interface between bactofilin-mediated membrane organization, DNA binding, and the response to cell wall stress. This could represent a novel switching mechanism by which B. subtilis can flexibly respond to structural challenges.