Interacting bactofilins impact cell shape of the MreB-less multicellular Rhodomicrobium vannielii

Abstract

Most non-spherical bacteria rely on the actin-like MreB cytoskeleton to control synthesis of a cell-shaping and primarily rod-like cell wall. Diverging from simple rod shape generally requires accessory cytoskeletal elements, which locally interfere with the MreB-guided cell wall synthesis. Conserved and widespread representatives of this accessory cytoskeleton are bactofilins that polymerize into static, non-polar bundles of filaments. Intriguingly, many species of the Actinobacteria and Rhizobiales manage to grow rod-like without MreB by tip extension, yet some of them still possess bactofilin genes, whose function in cell morphogenesis is unknown. An intricate representative of these tip-growing bacteria is Rhodomicrobium vannielii; a member of the hitherto genetically not tractable and poorly studied Hyphomicrobiaceae within the MreB-less Rhizobiales order. R. vannielii displays complex asymmetric cell shapes and differentiation patterns including filamentous hyphae to produce offspring and to build dendritic multicellular arrays. Here, we introduce techniques to genetically access R. vannielii, and we elucidate the role of bactofilins in its sophisticated morphogenesis. By targeted mutagenesis and fluorescence microscopy, protein interaction studies and peptidoglycan incorporation analysis we show that the R. vannielii bactofilins are associated with the hyphal growth zones and that one of them is essential to form proper hyphae. Another paralog is suggested to represent a novel hybrid and co-polymerizing bactofilin. Notably, we present R. vannielii as a powerful new model to understand prokaryotic cell development and control of multipolar cell growth in the absence of the conserved cytoskeletal element, MreB.

Complete genome sequencing and annotation of Rhodomicrobium vannielii strain DSM166 suggest affiliation to Rhodomicrobium lacus

“…Multicellularity and differentiation capabilities suggest complex gene expression and regulation patterns. A genetic system for strain DSM166 has been developed recently ( 8 ).…”

Membrane binding properties of the cytoskeletal protein bactofilin

“…Bactofilin homologs are widespread among bacteria and involved in diverse cellular functions. In many species, they have important roles in cell shape determination, including the establishment or adjustment of cell curvature in Helicobacter pylori (Sycuro et al, 2010), Campylobacter jejuni (Frirdich et al, 2023), Leptospira biplexa (Jackson et al, 2018) and Rhodospirillum rubrum (Pöhl et al, 2024), the modulation of cell size in Chlamydia trachomatis (Brockett et al, 2021), the formation of buds in Hyphomonas neptunium (Pöhl et al, 2024), the stabilization of rod shape in Myxococcus xanthus (Koch et al, 2011) and Proteus mirabilis (Hay et al, 1999) and the synthesis of stalk-like cellular extensions in C. crescentus (Kühn et al, 2010), Asticcacaulis biprosthecum (Caccamo et al, 2020) and Rhodomicrobium vannielii (Richter et al, 2023). Other functions include DNA organization in M. xanthus (Anand et al, 2020;Lin et al, 2017) as well as cell motility in Bacillus subtilis (El Andari et al, 2015).…”