Defining the minimal structural requirements of DivIVA in filamentous Actinomycetota

Abstract

The morphogenetic protein DivIVA exhibits diverse functions across bacterial phyla. In Bacillota, DivIVA is primarily involved in cell division, whereas in Actinomycetota, it plays a central role in coordinating polar growth. Due to its essential nature, gaining insight into DivIVA function is challenging. Here we report on the functionality of truncated DivIVA proteins, using a unique divIVA deletion mutant created in cell wall-deficient Kitasatospora viridifaciens L-forms. DivIVA comprises an N-terminal domain, two coiled-coil regions separated by an intercoil linker, and a C-terminal domain. Deleting either the intercoil or the C-terminal region impacted branching dynamics. We also created a minimized variant wherein both were deleted simultaneously, containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. Expression of this minimized variant resulted in severe growth defects. Cells exhibited a strong increase in hyphal width and cell wall thickness, accompanied by frequent tip bursting. Finally, we successfully introduced chimeric DivIVA from the unicellular actinobacterium Mycolicibacterium smegmatis with an N-terminal domain of Kitasatospora viridifaciens, demonstrating functional conservation within the phylum. In contrast, a chimeric DivIVA from Bacillus subtilis could not support growth, underscoring that polar growth is encoded within Actinomycetota-specific amino acid motifs encoded in the first and second coiled-coil. These findings enhance our understanding of the structure-function relationship for DivIVA and present new opportunities to study polar growth. ImpactDivIVA is essential for polar growth in Actinomycetota. In Streptomycetaceae, this membrane-binding protein localizes at growing hyphal tips and along lateral hyphal walls where new branches emerge. Due to its essentiality, the structural relationship of DivIVA between unicellular and multicellular species remains elusive. Using a Kitasatospora viridifaciens L-form divIVA deletion mutant, we expressed truncated DivIVA variants to identify essential regions. Deleting two large unstructured domains produced a minimized variant containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. This strongly impacted morphogenesis, increasing hyphal width and cell wall thickness, and leading to hyphal tip bursting. Finally, we successfully substituted DivIVA of K. viridifaciens with that from Mycolicibacterium smegmatis. Furthermore, Bacillus subtilis DivIVA could not facilitate reversion, showing that polar growth depends on amino acid motifs unique to Actinomycetota. These findings enhance our understanding of the structure-function relationship of DivIVA and offer new opportunities to study polar growth.