Biology subjects

Flardh, K.

Publications and source records attributed to Flardh, K..

3 recordsLinked to original sources

bioRxiv2025

Defining the minimal structural requirements of DivIVA in filamentous Actinomycetota

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.

microbiology↗

bioRxiv2021

Protein phosphatase SppA regulates apical growth and dephosphorylates cell polarity determinant DivIVA in Streptomyces coelicolor

Bacteria that exhibit polar growth, i.e. build their peptidoglycan cell walls in restricted zones at cell poles, often show large morphological diversity and plasticity. However, their mechanisms for regulation of cell shape and cell wall assembly are poorly understood. The Gram-positive Streptomyces bacteria, like other Actinobacteria, depend on the essential coiled coil protein DivIVA for establishment of cell polarity and direction of polar growth. Streptomycetes grow as filamentous hyphae that exhibit tip extension. New hyphal tips are generated by lateral branching. Cell shape is largely determined by the control of cell wall growth at these hyphal tips. The Ser/Thr protein kinase AfsK is involved in controlling polar growth and directly phosphorylates DivIVA. Here, we identify a protein phosphatase in Streptomyces coelicolor, SppA, that dephosphorylates DivIVA in vivo and in vitro and affects growth and cell shape. An sppA mutant shows reduced rate of hyphal tip extension, altered hyphal branching patterns, and exhibits frequent spontaneous hyphal growth arrests, all contributing to the unusually dense mycelial structure and slow growth rate that characterize sppA mutants. These phenotypes are largely suppressed in an afsK sppA double mutant, showing that AfsK and SppA partially affect the same regulatory pathway and share target proteins that are involved control of polar growth in S. coelicolor. Strains with a non-phosphorylatable mutant DivIVA were constructed and confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. Altogether, this study identifies a PPP-family protein phosphatase directly involved in the control of polar growth and cell shape determination in S. coelicolor and underscore the importance of eukaryotic-type Ser/Thr phosphorylation in regulation of growth and cell envelope biogenesis in Actinobacteria. Author summaryGram-positive bacteria often use eukaryotic-like Ser/Thr protein phosphorylation in the regulation of central processes related to growth and cell cycle progression. Members of the Actinobacteria, including pathogenic mycobacteria and antibiotic-producing streptomycetes, exhibit a distinctive mode of polar growth, with cell wall synthesis being restricted to zones at cell poles and directed by the essential cell polarity determinant DivIVA. Previously, we have shown that Streptomyces coelicolor modulates its machinery for polar growth and cell shape determination via the Ser/Thr kinase AfsK, which phosphorylates DivIVA. Here, we identify a phosphoprotein phosphatase that targets DivIVA and reverses the AfsK-mediated phosphorylation. We show that this phosphatase has strong effects on polar growth and cell shape, including hyphal branching and the rate of tip extension. The effects are to a large extent dependent on the kinase AfsK but do not require DivIVA phosphorylation as such. The results identify a new regulator of polar growth in streptomycetes, and highlight the importance of Ser/Thr phosphorylation in regulation of actinobacterial growth and cell shape determination.

microbiology↗

bioRxiv2020

The Streptomyces volatile 3-octanone alters auxin/cytokinin and growth in Arabidopsis thaliana via the gene family KISS ME DEADLY

Plants enhance their growth in the presence of particular soil bacteria due to volatile compounds affecting the homeostasis of plant growth hormones. However, the mechanisms of volatile compound signaling and plant perception has been unclear. This study identifies the bioactive volatile 3-octanone as a plant growth stimulating volatile, constitutively emitted by the soil bacterium Streptomyces coelicolor grown on a rich medium. When 3-octanone is applied to developing Arabidopsis thaliana seedlings, a family-wide induction of the Kelch-repeat F-box genes known as KISS ME DEADLY (KMD) subsequently alters auxin/cytokinin homeostasis to promote the growth of lateral roots and inhibit the primary root. Loss of function of the KMD family or other alterations of auxin/cytokinin homeostasis suppresses the volatile-induced growth response. This reveals a function of KMDs in the pathway of microbial volatile perception and plant growth responses. Significance StatementVolatiles from soil microbes are profound stimulators of plant growth. This work identifies for the first time a plant hormone signaling regulator, the gene family KISS ME DEADLY (KMD), to be an early essential step in plant growth promotion by a soil bacterial volatile, 3-octanone. The KMD-regulated gene network alters the tissue sensitivity balance for the growth hormones auxin and cytokinin, modifying root growth rate and architecture. Previously, the Kelch repeat F-box gene family of KMDs have been shown to be important down-regulators of both positive cytokinin signaling and phenylpropanoid biosynthesis, but upstream cues were unknown. This report places the KMD family regulation of plant growth and defense into its biotic context.

plant biology↗