Filamentous growth, cell envelope architecture, and surface appendages of a member of the Chloroflexota, Litorilinea aerophila

Litorilinea aerophila, a filamentous bacterium of the phylum Chloroflexota (class Caldilineae), exhibits unique morphological and cell envelope features that challenge traditional bacterial models. Initially described as Gram-negative, Chloroflexota are increasingly considered as monoderm, lacking a true outer membrane. In this study, we investigated the growth behaviour, cell morphology, and cell appendages of Litorilinea aerophila using light microscopy, cryo-electron tomography, and structural biology. Dry weight-based growth assays revealed a prolonged lag phase (~40 h) followed by exponential and stationary phases. Light and fluorescence microscopy revealed irregular indentations along cell filaments, accompanied by a diffuse distribution of DNA, indicating a multicellular organization. Thin-section electron microscopy confirmed septa formation, and in late growth stages, filaments became shorter with more defined indentations and membrane vesicle release. Next to the already characterised bacterial archaellum of Litorilinea aerophila, two additional types of surface appendages were identified: (i) pilus-like structures consistent with Tad pili, and (ii) grappling hook like structures. These findings contribute to the understanding of cell envelope diversity, growth, and surface structures in filamentous Chloroflexota.