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Function of a novel SPFH domain protein in bacteria

Subject Area Metabolism, Biochemistry and Genetics of Microorganisms
Cell Biology
Term from 2018 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 414320409
 
Final Report Year 2025

Final Report Abstract

SPFH domain proteins, named after a conserved domain found in stomatin, prohibitin, flotillin, and HflK/C, represent a conserved family of membrane-associated proteins present across all domains of life. These proteins are typically localized at cellular membranes, either as integral or peripheral components. SPFH domain proteins are involved in diverse cellular functions including membrane organization, signal transduction, mitochondrial activity, and protein quality control. Genetic deletion of individual SPFH domain-containing genes often results in pleiotropic phenotypes, indicating their involvement in fundamental homeostatic processes that influence multiple molecular pathways. In the model organism Bacillus subtilis, two flotillin homologs, FloA and FloT, have been extensively characterized. Both proteins form large, membrane-associated, oligomeric complexes. Their expression is upregulated in response to envelope stress via the alternative sigma factor sigma W. Like eukaryotic flotillins, FloA and FloT are associated with detergentresistant membrane extractions. Although DRMs are a highly artificial extraction method and do not correspond to real membrane domains in vivo, they are thought to enrich proteins and lipids from ordered lipid phases. In agreement with DRM extraction, bacterial flotillins localize to membrane microdomains with reduced fluidity as shown by co-localization studies using lipid packing sensitive dyes. Here, we demonstrated using in vitro reconstitution of FloA and FloT into artificial membranes their capacity to reduce lipid order, reaching down to the terminal carbons of the acyl chains. Thus, SPFH domain proteins may act as membrane destabilizers that counteract Lo domain coalescence. Deletion of floA and floT leads to decreased membrane fluidity and severe impairment of membrane protein function, such as peptidoglycan synthesis. This phenotype can be rescued by membrane fluidizing agents, confirming the role of flotillins in regulating membrane physical properties. B. subtilis contains a third SPFH domain protein, encoded by ydjI, and renamed SamI due to its association with starvation and motility phenotypes in the course of this study. SamI (ydjI) is co-transcribed with pspA, a homolog of the eukaryotic ESCRT III complex, and two membrane proteins SamG (ydjG) and SamH (ydjH). SamI lacks a transmembrane domain and requires SamG and SamH for stable membrane association. It colocalizes with more fluid membrane domains and does not overlap with flotillin-defined microdomains. Upon envelope stress, SamI and PspA form multiple membrane associated foci dependent on sigma W activation. Deletion of samI causes a measurable decrease in membrane fluidity and induces a transcriptional response consistent with nutrient deprivation. RNA sequencing of surface-grown biofilm colonies revealed upregulation of genes involved in glycolysis and stress adaptation, and repression of those associated with gluconeogenesis, motility, and the methionine salvage pathway. These findings correlate with severe impairments in swimming and swarming motility in both domesticated and wild-type strains of B. subtilis. Biofilm formation is also disrupted in the absence of SamI, as indicated by smooth colony morphology and the loss of the characteristic proteinaceous surface layer formed by BslA. While bslA transcription is unaffected, translation is diminished, possibly due to stress-induced translational repression. Electron microscopy confirmed the absence of the extracellular matrix layer in samI mutants. These observations establish SamI as a critical factor integrating membrane state and environmental stress signals to modulate developmental programs in B. subtilis.

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