Mad28, a conserved actin-like protein in deep-branching magnetotactic bacteria, exhibits cell curvature-dependent...

Magnetotactic bacteria (MTB) synthesize magnetic bacterial organelles called magnetosomes, which enable them to navigate along the geomagnetic field in aquatic environments. The actin-like cytoskeletal protein MamK forms filaments that associate with magnetosomes and mediate their positioning. Interestingly, in seven phyla, including Desulfobacterota, MTB encodes a second actin-like protein, Mad28, alongside MamK, within the magnetosome island-a genetic region responsible for magnetosome synthesis. In this study, we characterized the structure and function of this alternative magnetosome-associated cytoskeletal protein, Mad28. Magnetosome-specific localization of Mad28 in Solidesulfovibrio magneticus RS-1 was confirmed using immunoblotting, immunofluorescence microscopy, and correlative light and electron microscopy. To examine whether Mad28 and MamK have distinct or overlapping roles in magnetosome positioning, we tested the ability of Mad28^RS-1 or MamK^RS-1 to rescue the MamK-dependent static magnetosome positioning phenotype in Magnetospirillum magneticum AMB-1. Live-cell imaging revealed that MamK^RS-1 expression restored static magnetosome positioning, whereas Mad28^RS-1 expression had no effect, suggesting functional divergence between the two proteins. We further examined the potential role of Mad28 in sensing cellular geometry by comparing the localization of a Mad28-Dendra2 fusion protein in wild-type rod-shaped Escherichia coli and vibrio-shaped E. coli cells expressing Crescentin. Remarkably, Mad28 exhibited a curvature-dependent localization pattern in E. coli. These findings provide direct evidence that the actin-like protein Mad28 presents an affinity with membrane curvature in bacterial cells. In conclusion, the dual cytoskeletal systems-MamK and Mad28-contribute to magnetosome positioning through distinct mechanisms in deep-branching MTB.

Bacteria are capable of precisely positioning nanosized, membrane-enclosed organelles within their limited cellular spaces. This study shows that two distinct actin-like proteins contribute to magnetosome positioning through separate mechanisms in deep-branching magnetotactic bacteria. This contrasts with the evolutionary strategy observed in eukaryotic cells, where a single actin protein performs multiple functions. Furthermore, the findings suggest that the protein Mad28 is involved in sensing membrane curvature, introducing a novel functional property for bacterial actin-like proteins. These findings offer new insights into the role of the cytoskeleton in organelle positioning within micron-scale bacterial cells.