Cryo-Electron Tomography Reveals the Complex Ultrastructural Organization of Multicellular Filamentous Chloroflexota ( Chloroflexi ) Bacteria.

The cell biology ofChloroflexotais poorly studied. We applied cryo-focused ion beam milling and cryo-electron tomography to study the ultrastructural organization of thermophilicRoseiflexus castenholziiandChloroflexus aggregans, and mesophilic "Ca. Viridilinea mediisalina." These species represent the three main lineages within a group of multicellular filamentous anoxygenic phototrophicChloroflexotabacteria belonging to theChloroflexalesorder. We found surprising structural complexity in theChloroflexales. As with filamentous cyanobacteria, cells ofC. aggregansand "Ca. Viridilinea mediisalina" share the outer membrane-like layers of their intricate multilayer cell envelope. Additionally, cells ofR. castenholziiand "Ca. Viridilinea mediisalina" are connected by septal channels that resemble cyanobacterial septal junctions. All three strains possess long pili anchored close to cell-to-cell junctions, a morphological feature comparable to that observed in cyanobacteria. The cytoplasm of theChloroflexalesbacteria is crowded with intracellular organelles such as different types of storage granules, membrane vesicles, chlorosomes, gas vesicles, chemoreceptor-like arrays, and cytoplasmic filaments. We observed a higher level of complexity in the mesophilic strain compared to the thermophilic strains with regards to the composition of intracellular bodies and the organization of the cell envelope. The ultrastructural details that we describe in theseChloroflexalesbacteria will motivate further cell biological studies, given that the function and evolution of the many discovered morphological traits remain enigmatic in this diverse and widespread bacterial group.