The stomatin-like protein StlP organizes membrane microdomains to govern polar growth in filamentous actinobacteria under hyperosmotic stress

The stomatin-like protein StlP organizes membrane microdomains to govern polar growth in filamentous actinobacteria under hyperosmotic stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The stomatin-like protein StlP organizes membrane microdomains to govern polar growth in filamentous actinobacteria under hyperosmotic stress Dennis Claessen, Xiaobo Zhong, Sarah Baur, Veronique Ongenae, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3811693/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The cell wall represents an essential structure conserved among most bacteria, playing a crucial role in growth and development. While extensively studied model bacteria have provided insights into cell wall synthesis coordination, the mechanism governing polar growth in actinobacteria remains enigmatic. Here we identify the stomatin-like protein StlP as a pivotal factor essential for orchestrating polar growth in filamentous actinobacteria under hyperosmotic stress. StlP facilitates the establishment of a membrane microdomain with increased membrane fluidity, a process crucial for maintaining proper growth. The absence of StlP leads to branching of filaments, aberrant cell wall synthesis, thinning of the cell wall, and the extrusion of cell wall-deficient cells at hyphal tips. StlP interacts with key components of the apical glycan synthesis machinery, providing protection to filaments during apical growth. Introduction of StlP in actinobacteria lacking this protein enhances polar growth and resilience under hyperosmotic stress, accompanied by the formation of a membrane microdomain. Our findings imply that stomatin-like proteins, exemplified by StlP, confer a competitive advantage to actinobacteria encountering hyperosmotic stress. Given the widespread conservation of StlP in filamentous actinobacteria, our results propose that the mediation of polar growth through membrane microdomain formation is a conserved phenomenon in these bacteria. Biological sciences/Microbiology Biological sciences/Biochemistry membrane microdomain filamentous Actinobacteria streptomyces stomatin StlP polar growth cell wall-deficient cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The cell wall is considered an essential structure in bacteria that protects cells from environmental stresses 1,2 . To enable bacterial growth, the cell wall needs to be expanded, which involves inserting new cell wall material at the sites of growth. Elongation of rod-shaped cells typically occurs in two distinct manners 3 . Some rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis elongate by incorporating new cell wall material in a rather diffuse manner in the cylindrical part of the cell 4 . By contrast, other bacteria grow by inserting new cell wall material at the cell poles, referred to as polar growth, which is widespread in actinobacteria 5,6 . This mode-of-growth has been well studied in actinomycetes, which are filamentous bacteria that form branched mycelial networks in soil environments. In their natural environment, actinomycetes are often confronted with suboptimal conditions, such as fluctuations in water availability causing dramatic osmotic imbalances 7 . Paradoxically, we recently showed how conditions of hyperosmotic stress causes shedding of the cell wall in several actinomycetes, implying that such conditions interfere with the process of cell wall growth 8 . Polar growth in actinomycetes is guided by the cytoskeletal protein DivlVA, which localizes in actively growing tips acting as a scaffold for other proteins involved in organizing tip growth, such as the coiled-coil protein Scy and the intermediate filament-like protein FilP 9–11 . Unlike Scy and FilP, DivIVA plays an essential function in polar growth, by directly interacting with the machinery involved in synthesis of peptidoglycan, a major constituent of the cell wall 12,13 . The partial depletion of DivIVA caused hyphal bulging and irregular branching 9 . Scy is an unusual long coiled-coil protein that co-localizes with DivlVA at hyphal tips. Scy was suggested to form higher order assemblies and thereby serves as a hub to stabilize the tip-organizing center, which also includes cell division proteins and proteins involved in chromosome segregation 11,14,15 . Collectively, these proteins make sure that cell wall synthesis is coordinated with chromosome segregation to ensure proper growth and development of the mycelium. DivlVA also interacts with the putative cellulose synthase CslA 16 . The glycan produced by CslA is thought to provide protection to the tips, which are constantly remodeled during growth, in particular under conditions of osmotic stress 16 . Synthesis of the cellulose-like glycan not only depends on the synthase CslA, but also on a range of other proteins that are all encoded in a conserved gene cluster (Supplementary Fig. 1A). Together with CslA, the radical copper oxidase GlxA are the key proteins responsible for synthesis and likely modification of the glycan 17,18 . Following synthesis of the glycan, the lytic polysaccharides monooxygenase LpmP and endoglucanase CslZ facilitate deposition of the glycan chain at the cell surface, possibly by creating a passage through the thick peptidoglycan (PG) layer 19 . The cooperation of CslA/GlxA and CslZ/LpmP implies that a multicomplex is established at the tip related to the proper synthesis and secretion of the cellulose-like glycan. One of the proteins in the cellulose biosynthetic gene cluster that has not been studied encodes a stomatin/prohibitin/flotillin/HflK/C (SPFH)-domain protein, hereinafter referred to as StlP (for stomatin-like protein). The SPFH-superfamily proteins facilitate formation of lipid rafts, which in eukaryotes often contain protein complexes that collectively carry out important biological processes, such as ion channel regulation and touch sensation 20,21 . Notably, prokaryotic SPFH proteins, such as flotillins, also form comparable structures known as functional membrane microdomains (FMMs) 22 . These FMMs were shown to be involved in cell wall biosynthesis in B. subtilis 23 and Staphylococcus aureus 24 . Additionally, it was revealed that, in B. subtilis , flotillins play a direct role in controlling membrane fluidity homeostasis 23 , with their expression intriguingly regulated by stress-specific signals 25 . In this study, we reveal the pivotal role of StlP in coordinating the creation of a microdomain crucial for sustaining tip growth in Streptomyces coelicolor during hyperosmotic stress conditions. The absence of StlP leads to anomalous hyphal shape alterations and, notably, the expulsion of cells lacking their cell wall. StlP undergoes polymerization into oligomers and localizes within the membrane, culminating in the formation of a membrane region characterized by heightened fluidity. This phenomenon is essential for harmonizing the growth of both membrane and cell wall during tip extension. Significantly, the ectopic expression of StlP in actinomycetes, known for naturally extruding wall-deficient cells, effectively prevents such extrusion. These findings collectively underscore the significance of StlP in establishing a membrane microdomain at hyphal tips, thereby playing a critical role in facilitating proper cell wall assembly under hyperosmotic stress conditions in filamentous actinobacteria. Results StlP is a stomatin-like protein in Streptomyces coelicolor StlP is encoded in the conserved cellulose biosynthesis gene cluster in streptomycetes and located downstream of lpmP 19 (Supplementary Fig. 1A). Analysis of protein domains with InterPro and structure prediction via AlphaFold reveal that StlP contains three domains: a disordered region (aa 1-100), followed by a transmembrane hairpin composed by two helices (aa 106–126 and 149–168), and a SPFH (stomatin, prohibitin, flotillin, HflK/C) domain (aa 209–311) (Supplementary Fig. 1B, Supplementary Fig. 1C). Considering the diverse membrane topologies observed in members of the SFPH superfamily 26 , we examined the membrane topology of StlP to determine its classification within the family of SPFH-containing proteins. In silico analyses predicted that the C-terminal SPFH domain of StlP is in the cytoplasm (Supplementary Fig. 1D). To validate this prediction, we employed a β-lactamase assay in which this enzyme was fused to the C-terminus of StlP as described 27 . E. coli cells expressing StlP-BlaM NS fusions demonstrate sensitivity to ampicillin, in contrast to cells expressing BlaM FL with its original signal peptide (Supplementary Fig. 1E). This result confirms that the C-terminal SPFH domain of StlP is situated in the cytoplasm, consistent with the observed membrane topology in podocin/stomatin family proteins 20 and its N-terminal transmembrane hairpin. In contrast to StlP, other SPFH-like proteins from S. coelicolor have a significantly different predicted membrane topology (Supplementary Fig. 2). A phylogenetic analysis, utilizing amino acid sequences from well-known prokaryotic and eukaryotic SPFH proteins, revealed that S. coelicolor StlP and Mouse stomatin STOML-1 form a monophyletic clade, indicating that StlP resembles a stomatin-like protein (Supplementary Fig. 3A). Further sequence alignment of StlP with other stomatins indicates that Streptomyces StlP contains the signature proline 28 residue required for membrane hairpin formation and a comparable domain arrangement (Supplementary Fig. 3B). This alignment suggests that StlP shares more similarities with eukaryotic stomatins than prokaryotic stomatins. In summary, these findings collectively establish StlP as a stomatin-like protein within the prokaryotic family of SPFH proteins. StlP is important for morphogenesis under osmotic stress conditions To investigate the function of StlP, a stlP mutant was created that contained a Tn5062 transposon insertion, positioned upstream of the stomatin domain. This constructed stlP mutant was used to study its phenotype in various conditions. The stlP mutant displayed significantly reduced growth rates in both TSBS and LPB liquid medium, which contain 10 and 22% sucrose, respectively (Fig. 1 A and Supplementary Fig. 4A). On MS agar medium (without sucrose), no apparent differences in growth were observed between the parent and the stlP mutant (Supplementary Fig. 4B). By contrast, when grown on LPMA agar medium (containing 22% sucrose), the average diameter of individual colonies of the stlP mutant (1.4 ± 0.3 mm) was significantly reduced compared to the wild-type strain (3.8 ± 0.6 mm) (Fig. 1 B, Fig. 1 C), suggesting that growth was severely hampered. Furthermore, we noticed that excess membrane was extruded from hyphal tips of the stlP mutant, as evident in the FM5-95 panels comparing M145 and ∆ stlP in Fig. 1 C. Additionally, we observed many DNA-containing vesicles present in the medium (Fig. 1 D) that were reminiscent of cell wall-deficient cells extruded by several filamentous actinobacteria. These vesicles were absent in the parental strain, consistent with earlier findings 8 . Time-lapse imaging revealed that the vesicles were extruded from hyphal tips (Fig. 1 E, Supplementary Movies 2). Quantification revealed that the mutant formed 2.5x10 5 vesicles ml − 1 , while none were found in the parental strain (Supplementary Fig. 5). To confirm that these phenotypes were caused by the absence of StlP, we introduced plasmid pXZ15 in the mutant, in which stlP is expressed from the constitutive gapAp promoter. Reintroduction of this plasmid partially restored the growth speed in liquid media (Fig. 1 A, Supplementary Fig. 4A) and the average colony diameter on LPMA medium (2.5 ± 0.5 mm) (Fig. 1 B, Fig. 1 C), while extrusion of membranes and DNA-containing vesicles was also reduced by 80% (Fig. 1 D, Supplementary Fig. 5). Microscopy analysis also indicated that the hyphal branching pattern was affected by the deletion of stlP (Fig. 1 F, Supplementary Fig. 4D, Supplementary Movies 1 and 2). More specifically, the number of branches was dramatically increased in the stlP mutant when grown under hyperosmotic stress conditions (LPMA medium) (Fig. 1 F). To quantitatively compare branching between the strains, we measured the distance from the tip to the proximal branching point in hyphae (Supplementary Fig. 4D). Deletion of stlP changed the distribution of the tip-to-branch distances significantly. We found that more than 60% of all hyphae in ∆ stlP had a proximal branch within the first 5 µm from the tip (Fig. 1 G), as compared to approximately 15% for the parent and the complemented mutant. Also, no hyphae of the stlP mutant branched further than 35 µm from the tip. Additionally, when grown on MS medium, hyphae with a tip-to-branch distance less than 10 µm accounted for 3.6% and 8.9% of the parent and mutant strain, respectively (Supplementary Fig. 4E). This suggests a mild impact on the branching pattern due to the deletion of stlP under normal growth conditions. Taken together, these findings underscore that the absence of stlP significantly influences the growth and morphogenesis of S. coelicolor , particularly manifesting under conditions of hyperosmotic stress. StlP is important for spatially confining cell wall synthesis to hyphal tips To investigate the localization of StlP, we introduced pXZ16 into S. coelicolor M145, thereby constitutively expressing a C-terminal mCherry fusion to StlP. Foci of StlP-mCherry mostly localized at growing tips and emerging branches (Fig. 2 A). We also localized the polar growth determinant DivIVA in the presence and absence of StlP by expressing a DivIVA-mCherry fusion from the constitutive gapAp promoter. Interestingly, in the absence of StlP, DivIVA-mCherry was not only localized to hyphal tips but was also found in numerous foci along the cylindrical part of the filaments (Fig. 2 B), suggesting the cell wall synthesis was no longer confined to the apex. In agreement, nascent PG was incorporated at multiple sites along the filament in the stlP mutant (Fig. 2 C), coinciding with an increase in the average diameter of the hyphae (Fig. 2 D). Furthermore, we noticed that multiple synthesis foci of the cellulose-like glycan by CslA appeared at established and emerging hyphal tips, which is contrary to the parental strain (Fig. 2 E). Deposition of the cellulose-like glycan was affected in the stlP mutant and was no longer spatially confined to the hyphal tip (Fig. 2 E). Quantitative analysis indicated that the absence of StlP let to some 60% reduced accumulation of glycans at hyphal tips, indicated by calcofluor white staining (Fig. 3 F). Furthermore, the reduced glycan levels made the stlP mutant sensitive to lysozyme (Fig. 3 G), which was previously observed in other mutants affected in apical glycan deposition. Glycan deposition and lysozyme resistance were restored when the complementation plasmid pXZ15 was introduced in the stlP mutant (Fig. 3 E, Fig. 3 F and Fig. 3 G). To accurately evaluate the effect of the absence StlP on cell wall thickness of S. coelicolor , we conducted a thorough examination by preparing and imaging sacculi of M145 and the ∆ stlP strains using cryo-electron tomography (cryo-ET). The analysis of cell wall thickness measurement revealed a significant reduction in the overall cell wall thickness upon stlP deletion compared to the parental strain. Specifically, the thickness in the apical region decreased from 35.5 ± 1.7 nm to 6.4 ± 0.6 nm, while the thickness in the subapical region was reduced from 37.1 ± 2.3 nm to 9.9 ± 0.6 nm (Fig. 2 H and 2 I). These results demonstrate the importance of StlP for cell wall synthesis and thickness of S. coelicolor . To establish how StlP contributes to delocalized cell wall synthesis, we tested interactions of StlP with the proteins involved in tip growth and cellulose biosynthesis. To this end, constructs were generated that produced C-terminal fusions of StlP, LpmP, SCO2835, CslA, GlxA, CslZ, DivIVA, Scy and FilP to either the T25 or T18 fragments of the adenylate cyclase, respectively. Co-transformation of these constructs in E. coli BTH101 revealed that StlP robustly interacts with LpmP, SCO2835, CslA and CslZ, but also with itself and weakly interacts with GlxA (Supplementary Fig. 6). Furthermore, StlP did not interact with DivIVA, Scy or FilP (Supplementary Fig. 6). Thus, StlP directly interacts with components of the cellulose biosynthesis complex and spatially confines cell wall synthesis to hyphal tips in S. coelicolor . Deletion of stlP induces cell death in S. coelicolor We noticed a significant abundance of contractile injection system (CIS) structures within the sacculi of the stlP mutant cultivated for 16 h, in contrast to the absence of these structures in the sacculi of an equivalently aged parental strain (Fig. 2 H). These CIS structures were recently found to be involved in programmed cell death regulation 29,30 . The observation of CIS led us to investigate if the deletion of stlP increased cell death of S. coelicolor . To investigate this, M145 and its stlP mutant were grown for 16 h on LPMA medium. Cell death in the mycelia was measured using a bacterial viability assay 31 (see Materials and Methods). In principle, when SYTO9 and propidium iodide (PI) nucleic acid stains are present simultaneously, stained viable mycelia and dead mycelia exhibited green, and red fluorescence, respectively. The live/dead (SYTO9/PI) ratio was notably reduced in the stlP mutant when compared to the parent strain (see Supplementary Fig. 8). This decrease suggests increased cell death in the stlP mutant, aligning with our findings that the deletion of stlP significantly impeded the growth of liquid-cultured S. coelicolor (Fig. 1 A, supplementary Fig. 4A) Altogether, these results imply that deletion of stlP contributes to increased cell death of S. coelicolor . StlP oligomerizes and forms membrane microdomain at hyphal tips of S. coelicolor Bacterial two-hybrid analysis suggested that StlP interacts with itself but also with components of the machinery involved in glycan synthesis (Supplementary Fig. 6). To verify if StlP interacts with itself via the stomatin domains, the corresponding domain (aa 204–326; referred to as StlP SD ) was expressed in E. coli BL21(DE3). The StlP SD monomer was expressed, showing a predicted molecular mass of 18 kDa (lane BC, Fig. 3 A). When samples were concentrated to 1 mg ml − 1 , a ladder of oligomers was found (lane AC), suggesting that monomers can self-assemble. Notably, less monomers and more crosslinked highly ordered polymers were observed with increasing concentrations of the cross-linker glutaraldehyde (Fig. 3 A). These results suggest that StlP can assemble into oligomers via its stomatin domain at high concentrations, which in the full-length protein would probably result in formation of a membrane microdomain via its two N-terminal transmembrane helices. To corroborate this hypothesis, the partial structure of SltP (aa 106–326), which covers the two N-terminal TMHs (aa 106–126 and aa 149–168) and the stomatin domain (aa 209–311) was predicted using AlphaFold 2.0. Given that the putative stomatin domain of FliL from the marine bacterium Vibrio alginolyticus , the only structure known of a bacterial stomatin, assembles into a symmetric ring consisting of 10 monomers, 10 StlP monomers were used for prediction of the structure. This predicted that the cytoplasmic stomatin domain of StlP assembles into a highly ordered multimer via an end-to-end interaction pattern, in which the N-terminal transmembrane helical hairpins of the monomers contribute to forming a symmetric ring (Fig. 3 B). In bacteria, SPFH proteins are involved in membrane fluidity homeostasis, evidenced by FloT and FloA that fluidize the membrane and absence of them leads to an overall rigidification 23,32 . We therefore predicted that the oligomerization of StlP leads to the formation of a local membrane r egion with i ncreased f luidity (RIF) at hyphal tips. To verify this prediction, we stained mycelia of S. coelicolor with DilC 12 , a lipid dye with high specificity for fluid membranes due to its short hydrocarbon tail 33,34 . We observed bright DilC 12 staining RIFs at hyphal tips, and also along hyphae grown for 16 h in LPB medium (Fig. 3 C). The signal corresponding to DilC 12 staining was absent from hyphal tips of the stlP mutant and restored in the complemented strain (Fig. 3 C). These results suggest that hyphal tips possess a RIF, which is dependent on the presence of StlP. To substantiate the presence of RIFs at hyphal tips, we conducted a quantitative assessment of membrane fluidity using the membrane-intercalating dye Laurdan. This dye exhibits a shift in fluorescence emission wavelength based on membrane fluidity 33,35 . After culturing mycelia of M145 and its stlP mutant in LPB medium for 16 hours, we stained them with Laurdan and then calculated the general polarization (GP) value at the tip region of each hypha (see Materials and Methods). In the absence of stlP , the average GP value (-0.09 ± 0.08) increased compared to its parental strain (-0.12 ± 0.13), inferring a decreased membrane fluidity in the stlP mutant (Fig. 3 D and Fig. 3 E). Furthermore, the apical membrane fluidity of the stlP mutant could be restored by raising the growth temperature to 37 ⁰C (Fig. 4 F), which aligns with the idea that elevated temperatures contribute to membrane phase transitions, leading to an increased fluidity 36 . Altogether, these results reveal that StlP oligomerizes and forms membrane domains with enhanced membrane fluidity at hyphal tips of S. coelicolor . Morphogenesis controlled by StlP is conserved in filamentous actinobacteria Our results indicate that StlP contributes to proper cell wall synthesis and membrane organization under hyperosmotic stress, and thereby controls morphogenesis of S. coelicolor . To see how prevalent StlP is, we combined PSI-BLAST and TMHMM prediction to identify stomatin homologues of StlP in the dataset of 15045 RefSeq representative bacteria and archaea. This analysis showed that the majority of StlP homologs are present in actinobacteria, while a few are also present in Rhizobium (Fig. 4 ). Furthermore, inside the filamentous actinobacteria, StlP orthologs are present in genera including Streptomyces, Kitasatospora and Streptacidiphillus (Fig. 4 ). In some clades of these bacteria, all members have an orthologue of StlP, while in others StlP is less common or virtually absent. Notably, Kitasatospora viridifaciens DSM40239 is among the clades that lack StlP (Fig. 4 ). To see how widespread the function of StlP is in other actinobacteria, the construct pXZ15, wherein the stlP was expressed from the constitutive gapAp promoter, was introduced into Kitasatospora viridifaciens DSM40239 via conjugation, which is known to extrude wall-deficient cells under hyperosmotic stress 8,37 . Importantly, constitutive expression of stlP induced the formation of a fluid membrane microdomain at hyphal tips in K. viridifaciens , as evidenced by DilC 12 staining (Fig. 5 A). Furthermore, constitutive expression of stlP significantly increased the average membrane fluidity of hyphae (Fig. 5 B, 5 C) and allowed colonies to cope much better with hyperosmotic stress, as shown by the strongly increased colony diameter of the strain expressing stlP (2.75 ± 0.6 mm) as compared to the parental strain (1.25 ± 0.9 mm) and by the reduced lateral branching of K. viridifaciens (Fig. 5 D, 5 E). Furthermore, constitutive expression of stlP largely abolished the extrusion of wall-deficient cells in K. viridifaciens (Fig. 5 F). By contrast, the constitutive expression of the HflK/C-like protein BOQ63_030050 in K. viridifaciens , which also belongs to the SPFH superfamily of proteins and shares 21% identity with StlP had no effect on the extrusion of wall-deficient cells (Supplementary Fig. 9, 10). These results demonstrate that StlP controls growth of filamentous actinobacteria under hyperosmotic stress by the localized control of membrane fluidity. Discussion Stomatin-like proteins are ubiquitous in all domains of life. A universal feature of these proteins is to form functional nanoscale microdomains in biological membranes. In turn, these microdomains serve as platforms to locate protein complexes involved in important biological processes, such as transducing mechanosensory signals in mice and the modulation of ion channels in mammalian cells 38–40 . The recent structural analysis of the stomatin-like protein FliL from Vibrio alginolyticus showed that FliL shares some structural elements with eukaryotic stomatins, suggesting that stomatin-like proteins are conserved from mammals to bacteria 40 . Here, we identified the stomatin-like protein StlP as a novel polar growth determinant in filamentous actinobacteria. We show that StlP is crucial for spatially organizing cell wall synthesis at hyphal tips in conditions of hyperosmotic stress, which likely provides competitive benefit to microbes frequently exposed to such conditions. Evolutionary investigation showed the group of stomatins are ancient and their evolution likely occurred at an early stage in the evolution of prokaryotes 41 . Most prokaryotic stomatins (so-called p-stomatins) have been found in archaeal species, for instance in isolates from environments with high temperatures ( Pyrococcus horikoshii , Archaeoglobus fulgidus and Methanothermobacter thermautotrophicus ) and isolates from sediments near the sea ( Aeropyrum pernix ) 41,42 . Some p-stomatins exist in bacterial species, such as in Photobacterium aphoticum isolated from coastal water 43 and the marine bacterium Vibrio alginolyticus 40 . However, their roles have not been characterized well. Excitingly, StlP, the p-stomatin of S. coelicolor , appears to play a pivotal role in regulating tip growth in conditions of hyperosmotic stress. Interestingly, S. coelicolor was originally isolated from the beach, which makes it plausible that S. coelicolor , contrary to K. viridifaciens isolated from mountain soil, is better adapted to salt-rich environments, for instance by having StlP. Stomatins oligomerize in cell membranes through interactions between their stomatin-domains, thus providing a scaffold for numerous other proteins. This functionality has been demonstrated for several stomatins, including mouse stomatin 39 , Pyrococcus horikoshii stomatin 44 and the stomatin FliL of Vibrio alginolyticus 40 . Cross-linking studies and structure predictions demonstrate that the stomatin domain of StlP oligomerizes and that StlP interacts with the machinery involved in synthesis of a cellulose-like glycan. One of these proteins is the cellulose synthase-like protein CslA, which in turn directly interacts with DivIVA 16 . Interestingly, PG synthesis of actinomycetes is directed at hyphal tips in a DivIVA-dependent manner. More specifically, DivIVA recruits penicillin-binding proteins (PBPs) during polarized growth, as deduced from the detected interaction between DivIVA and PBP3 in M. tuberculosis 11,12 . StlP seems to contribute to establishing a localized region in the cellular membrane carrying crucial components of the so-called tip organizing center, which controls cell wall synthesis during the polar growth in filamentous actinobacteria (Fig. 6 ). Indeed, the observation of diffused PG and surface cellulose synthesis foci and dramatically reduced cell wall thickness of the stlP mutant indicated that PG synthesis is significantly weakened upon the deletion of stlP (Fig. 2 ). We thus propose a model for the cell wall synthetic machinery at hyphal tips when filamentous actinobacteria grow in hyperosmotic stress conditions. In this model, DivIVA guides the machinery responsible for peptidoglycan (PG) and surface cellulose-like glycan synthesis to the poles through interactions with CslA and PBPs. At these poles, the systems are restricted by the formation of StlP rings, which, in turn, induce localized membrane fluidization (Fig. 6 ). The synthesis of the surface cellulose-like glycan involves substantial redox reactions facilitated by the presence of LpmP and GlxA. Here, the StlP ring, may serve to restrict oxidative stress within the ring, shielding proteins outside the StlP from this stress. Meanwhile, the StlP ring enables these filamentous actinobacteria to produce robust cell walls during polarized growth under conditions of hyperosmotic stress. Without StlP, the membrane fluidity in the tip region decreases. Combined with the loss of localized cell wall synthesis, this leads to the extrusion of wall-deficient cells. Reversely, constitutive expression of StlP in species that naturally lack this protein prevents the typical extrusion of wall-deficient cells. Taken together, this work provides a plausible mechanism how wall-deficient cells are extruded in polar-growing actinobacteria. By interrupting cell wall synthesis, by exposing the cells to hyperosmotic stress or antibiotics, the coordinated balance between membrane and cell wall synthesis is lost. In turn, this leads to shedding of excess membranes from the polar growth sites and is facilitated by the localized weakening of the cell wall. In summary, our work for the first time characterizes a stomatin-like protein that regulates tip growth in conditions of hyperosmotic stress. The strong phenotype associated with its absence make this also an interesting candidate to target in pathogenic bacteria that grow from the cell poles. In this context, it is noteworthy that mycobacteria have the ability to adopt a wall-deficient lifestyle 45 , and it’s worth highlighting that Mycobacterium tuberculosis possesses a StlP homolog. Material and methods Strains and growth conditions Strains used in this study are listed in Supplementary Table 1. Solid MS (Mannitol Soy flour) medium 46 was used for collection of Streptomyces spores and for conjugation experiments, while MYM medium 47 was used for obtaining Kitasatospora spores. To compare colony sizes and observe the release of cell wall-deficient (CWD) cells under hyperosmotic stress, solid LPMA medium 8 was used. TSBS 46 medium was used to grow Streptomyces in liquid medium without hyperosmotic stress. For quantification of the number of CWD cells, as well as measurement of hyphal diameters and membrane fluidity, liquid L-phase broth (LPB) was used 8 . Briefly, 10 6 CFU ml − 1 spores were inoculated in 20 ml LPB in 50 ml flasks without coil while shaking at 100 rpm ml − 1 . All Streptomyces and Kitasatospora strains were grown at 30°C. For hyphal branching detection, spores were inoculated onto cellophane membranes overlaying LPMA plates, which were then incubated at 30°C for 16 h prior to analysis. Lysozyme sensitive assay were performed essentially as described 19 . E. coli DH5α 48 was used for cloning and β-lactamase experiments. E. coli BL21 (DE3) 49 and BTH101 50 were used in protein expression and bacterial two-hybrid assays, respectively. For conjugation, E. coli ET12567 harboring pUZ8002 was used. All E. coli strains were grown in LB medium at 37°C with appropriate antibiotics, if needed. Plasmid construction and transformation Plasmids and primers used in this study are listed in Supplementary Table 2 and Supplementary Table 3, respectively. For complementation of the ∆ stlP mutant, the coding sequence of stlP (SCO2834) was amplified from genomic DNA of S. coelicolor with primers stlP-F/stlP-R1. The PCR product was ligated as an NdeI/BamHI fragment into plasmid hpXZ2 19 harboring the constitutive gapAp prompter of SCO1947, yielding plasmid pXZ15. For localization of StlP and DivlVA, the mCherry reporter was fused to the C-terminus of these two proteins. Coding sequences for stlP and divlVA were amplified from genomic DNA of S. coelicolor with primers stlP-F/stlP-R2 and 2077-F/2077-R, respectively. The gene encoding for mCherry was amplified from plasmid pGWS791 27 using primers mCh-F/mCh-R. After digestions with NdeI/HindIII (for stlP and divlVA ) and HindIII/XbaI (for mCherry ), combinations of stlP/mCherry and divlVA/mCherry were ligated with plasmid hpXZ2 that was cut with NdeI/XbaI, generating plasmid pXZ16 and pXZ17, respectively. To constitutively express the HflK/C-like protein BOQ63_030050 in Kitasatospora viridifaciens and the S. coelicolor stlP mutant, the coding sequence of BOQ63_030050 was amplified from genomic DNA of K. viridifaciens DSM40239 with primers 0050-F/0050-R. The PCR product was ligated as an NdeI/XbaI fragment into plasmid hpXZ2 harboring the constitutive gapAp prompter, yielding plasmid pXZ41. To express the stomatin-encoding domain of StlP (StlP SD ) in E. coli , nucleotides 610–1107 of stlP were amplified from S. coelicolor genomic DNA with primers 2834 sto -F/2834 sto -R and ligated into pET28a plasmid as a NdeI and HindIII fragment, yielding plasmid pXZ18. Consequently, StlP SD was expressed carrying a N-terminal Histidine-tag (6xHis). All plasmids were introduced into E. coli and Streptomyces via heat-shock transformation 51 and conjugation 46 , respectively. Inactivation of stlP in Streptomyces For inactivation of stlP in S. coelicolor and S. lividans , we used cosmid StE20 carrying the Tn5062 transposon inserted after nucleotide position 892 relative to the start site of stlP (kindly provided by Prof. Paul Dyson, see Supplementary Table 2). This cosmid was introduced into S. coelicolor via conjugation using ET12567/pUZ8002 46 , after which exconjugants were screened as described 52 . Colonies that were kanamycin-sensitive and apramycin-resistant were selected and used for further analysis. Mutants carrying the expected phenotype were verified by sequencing. Growth curve generation For preparing germinated spores, spores of different strains were resuspended in double-strength germination medium 46 at a final concentration of 10 6 CFU ml − 1 and incubated at 30 ⁰C for 6 ~ 8 h while shaking at 200 rpm min − 1 . For generating the biomass growth curve, the RoboLector L-4-BL-II equipped with a parallelized shaken cultivation device was used 53 . Briefly, the germinated spores were centrifuged and resuspended in either TSBS or LPB media before being distributed (1 ml per well) into a 48-well FlowerPlates (Basesweiler Germany). The temperature and humidity were set at 30 ⁰C and 85%, respectively. The biomass was collected automatically and measured in arbitrary units. All measurements were performed in triplicate. Bacterial 2-hybrid analysis To assess interactions of StlP with other tip-localizing proteins, the bacterial hybrid assay was used 50 . Therefore, lpmP (SCO2833), stlP (SCO2834), sco2835 , cslA (SCO2836), glxA (SCO2837), cslZ (SCO2838), divlVA (SCO2077), filP (SCO5396) and scy (SCO5397) were amplified using primers Th2833-F/Th2833-R, Th2834-F/Th2834-R, Th2835-F/Th2835-R, Th2836-F/Th2836-R, Th2837-F/Th2837-R, Th2838-F/Th2838-R, divlVA-F/divlVA-R, filP-F/filP-R and scy-F/scy-R, respectively. All amplified DNA fragments were cloned into the pKT25 and pUT18C plasmids using EcoRI and XbaI, yielding plasmids pXZ19 (pUT18C + lpmP ), pXZ20 (pKT25 + stlP ), pXZ21 (pUT18C + stlP ), pXZ22 (pKT25 + SCO2835 ), pXZ23 (pUT18C + SCO2835 ), pXZ24 (pKT25 + cslA ), pXZ25 (pUT18C + cslA ), pXZ26 (pKT25 + glxA ), pXZ27 (pUT18C + glxA ), pXZ28 (pKT25 + cslZ ), pXZ29 (pKT25 + divlVA ), pXZ30 (pUT18C + divlVA ), pXZ31 (pKT25 + filP ), pXZ32 (pUT18C + filP ), pXZ33 (pKT25 + scy ) and pXZ34 (pUT18C + scy ) (see Supplementary Table 2). E. coli BT101 carrying combinations of these constructs were used in bacterial 2-hybrid experiments to evaluate protein interactions as described 54 . Topology determination of StlP To study the transmembrane topology of StlP, the β-lactamase - encoding gene blaM without its signal sequence was fused to the 3’ end of stlP , as described previously 27 . Only if BlaM is secreted, cells will be resistant to ampicillin. To this end, a blaM variant lacking the region encoding the signal sequence for secretion ( blaM NS ) was amplified from plasmid pHJL401 55 with primers blaM-F/blaM-R. In parallel, full length blaM (including the region for the signal sequence, hereinafter referred to as blaM FL ) was amplified from the same plasmid using the blaM FL -F/blaM-R primers. The stlP gene was amplified from S. coelicolor genomic DNA using primers stlP-F/ stlP -R3. Subsequently, the amplified products were cut using restriction enzymes NdeI-HindIII ( stlP ), HindIII-EcoRI ( blaM NS ) and NdeI-EcoRI ( blaM FL ), and digested combinations of stlP / blaM NS , and blaM FL were separately ligated into pXZ2 19 that was cut with NdeI-EcoRI, yielding pXZ35 and pXZ36 (see Supplementary Table 2). E. coli DH5α harboring plasmid pSET152, pXZ35 and pXZ36 were used to assess the membrane topology of StlP, which were performed as described previously 27 . Full-length BlaM (BlaM FL ) expressed from pXZ36 served as a control for β-lactamase activity in the medium. Quantification of CWD cells Culturing and filtration of CWD cells of Streptomyces and Kitasatospora strains was essentially performed as described 8 , with the exception that S. coelicolor strains were grown for 16 h, while K. viridifaciens strains were grown for 40 h. Quantification of CWD cells was performed with a Bright-Line™ Hemocytometer (Merck), as described 56 . Briefly, 10 µl of filtered culture supernatant were loaded into the counting chamber, after which cells were quantified under a Zeiss Axio microscope equipped with an Axiocam 105 camera. CWD cell numbers were counted manually, and the density was calculated. For each strain, the measurements were performed in triplicate. Protein expression and purification To purify the stomatin-encoding domain of StlP (StlP SD ), E. coli BL21 (DE3) cells harboring plasmid pXZ17 were cultured at 37°C to an OD 600 of 0.8 in LB medium containing 50 mg ml − 1 kanamycin. Then, 0.5 mM isopropyl β-D-thiogalactopyranoside was added to induce protein expression, after which cells were grown at 30°C for 18 h. The induced cells were subsequently lysed by sonication in binding buffer (50 mM Tris–HCl, 200 mM NaCl, pH 8.0), and after centrifugation, the lysate was loaded on a Ni 2+ -chelating column equilibrated with binding buffer. Ten column volumes of washing buffer (50 mM Tris–HCl, 200 mM NaCl, 0.1 mM imidazole, pH 8.0) and 5 mL of elution buffer (50 mM Tris–HCl, 200 mM NaCl, 10 mM imidazole, pH 8.0) were used to wash and elute StlP SD , respectively. Finally, the protein was purified by gel filtration using a Hiload 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with buffer (50 mM Tris–HCl, 100 mM NaCl, pH 8.0). Sample fractions were analyzed on a 12.5% SDS-PAGE gel. Fractions were stored directly at -80°C or first concentrated to 1 mg ml − 1 with the 3 kDa molecular weight cutoff concentrator (Millipore) and then stored. Chemical cross-linking The cross-linking of StlP SD with glutaraldehyde (GA, 50% in H 2 O, Sigma) was performed as described 44 with the following modifications. Briefly, 1 mg ml − 1 of StlP SD was treated with 0.02, 0.05 or 0.1% GA for 10 or 60 min at room temperature in 50 µl buffer (50 mM Tris–HCl, 100 mM NaCl, pH 8.0). Reactions were quenched with 0.2 M glycine-NaOH (pH 9.5) for 5 min at room temperature. Detection of cross-linked protein was performed by loading the samples on a 12.5% SDS-PAGE gel. Microscopy To visualize the emergence of CWD cells, spores of the wild-type strain and the ∆ stlP mutant were pre-germinated in double strength germination medium 46 . Then 10 µl of germlings were used for live-imaging, for which an ibiTreat 35 mm low imaging dish (ibidi) and a LPMA-pad was used as before 8 . Live imaging was carried out using a Zeiss LSM900 Airyscan 2 microscope. If necessary, Z-Stack acquisitions were used. For visualization of membrane and nucleic acids, 0.05 mg ml − 1 FM5-95 and 0.5 µM SYTO-9 (Sigma) were used, respectively. The detection of nascent peptidoglycan was done by using BODIPY-FL vancomycin (Sigma), essentially as described 57 . Briefly, after growing Streptomyces strains in LPB medium for 16 h, mycelia were collected (3300 rpm min − 1 , 10 min) and resuspended in 200 µl of fresh LPB medium containing 1 µg ml − 1 BODIPY FL vancomycin and 1 µg ml − 1 vancomycin. After 10 min incubation at 30°C, the mycelia were washed 3 times with PBS. 5 µl of the washed mycelia was used for microscopy analysis using a Zeiss LSM900 Airyscan 2 microscope. For visualizing fluid membrane microdomains, mycelia were stained with DilC 12 33,34 . Briefly, DilC 12 was dissolved in DMSO and 10 mg ml − 1 stock was prepared. For sample preparation, spores of each strain were inoculated in LPB medium at a final concentration of 10 6 CFU ml − 1 . After 16h of growth, mycelia were collected by centrifuge (3300 rpm min − 1 , 10 min) and resuspended in prewarmed fresh LPB medium supplemented with 100 µg ml − 1 DilC12, followed by growth for 3 h at 30°C while shaking at 100 rpm min − 1 . Then, mycelia were collected, washed 3 times with prewarmed LPB supplemented with 1% DMSO, and resuspended in the same wash buffer. DilC 12 signal was detected via a Cy3 filter (535 nm excitation and 590 nm emission) using a Zeiss LSM900 Airyscan 2 microscope, wherein the cultivation chamber had been set 30°C to avoid temperature changes during imaging. For measurement of membrane fluidity, samples were prepared essentially as described 32 . Briefly, Laurdan (6-Dodecanoyl-N, N-dymethyl2-naphthylamine, Sigma) was dissolved in dimethylformamide (DMF) and a 10 mM stock was prepared. For preparation of mycelia for Laurdan staining, 20 ml 16 h-old mycelia were collected (3300 rpm min − 1 , 10 min) and resuspended in 1 ml 30°C pre-warmed LPB medium containing 1 mM Laurdan. For sample preparation of the stlP mutant, the culture was filtrated through a 100 µm cut-off filter (Falcon Cell Strainer 100 µm Nylon) to remove the majority of the CWD cells. The filtered mycelium was resuspended in 1 ml 30°C pre-warmed LPB medium and used for Laurdan staining. After 10 min incubation in the dark at 30°C, stained mycelia were collected again and washed twice in 30°C pre-warmed PBS buffer supplemented with 20% sucrose and 1% DMF, and finally resuspended in 200 µl pre-warmed PBS buffer supplemented with 20% sucrose. Fluorescent intensities were measured at 435 and 490 nm, following excitation at 350 nm using a Zeiss LSM900 Airyscan 2 microscope, wherein the cultivation chamber had been set 30°C to avoid temperature changes during imaging. To calculate the membrane fluidity at hyphal tips, the tip region was cropped as a square (1 µm x 1 µm) from the image and the corresponding generalized polarization (GP) value was determined as described 58 . For measurement of hyphal diameters, 16 h-old mycelia were collected (3300 rpm min − 1 , 10 min) and resuspended in fresh LPB medium containing 0.05 mg ml − 1 FM5-95. The distance between the stained membranes was used to measure the hyphal diameters by averaging the diameter at 3 distinct spots in the hypha. The cellulose-like glycan at hyphal tips was visualized by calcofluor white (Sigma) staining and quantified as previous described 19 . For analysis of hyphal branching patterns, mycelium was grown from single spore and imaged using a Zeiss Axio microscope equipped with an Axiocam 105 camera as described previously 19 . The distance from the tip to the proximal branch point was measured. A proximal branch was defined as having a length of 1–4 µm as previous described 59 . For measurement of colony sizes, strain were grown on LPMA medium in petri dishes with a 9 cm diameter aiming for ± 100 colonies per plate. After growing them for 5 ~ 7 days at 30°C, plates were scanned with Epson Perfection V37 scanner and colony size was measured subsequently. Mycelial Live/dead staining was performed using the LIVE/DEAD Bac Light™ Bacterial viability kit (L7012; ThermoFisher) following the manufacturer’s instructions. Briefly, spores of the wild-type strain and the stlP mutant were streaked on LPMA medium. After growth for 16h, the excised ager pieces were inverted and positioned atop 10 µl mixture of SYTO-9 and propidium iodide (PI) nucleic acid stains from the kit with final concentration at 5 µM. Images were taken using a Zeiss LSM900 Airyscan 2 microscope after 10 mins incubation. The viability was calculated by dividing the integrated grey intensity of the fluorescence in the green channel by the integrated intensity of the fluorescence in the red channel. All measurement and images processing were executed with ImageJ software (version 2.0.0/1.53c/Java 1.8.0_172/64-bit). Sacculus isolation and Cryo-electron tomography Isolation of sacculi of S. coelicolor M145 and the stlP mutant was essentially performed as described 60 , except that 16h-old liquid cultures were used and the step of removing teichoic acids was neglected. Sample preparation for cryo-electron tomography (cryo-ET) was performed as described 60 . Briefly, after adding the colloidal gold beads, sacculi solutions were vitrificated and applied on the EM grids. Grids were examined using a 120 kV Talos TEM (FEI/ThermoFisher) and cryo-ET data were collected using a Titan Krios instrument (ThermoFisher Scientific). The measurement of cell wall thickness was performed as described 60 . Bioinformatic analysis Protein domains and protein structures were predicted by InterPro ( https://www.ebi.ac.uk/interpro/ ) and AlphaFold 2.0 61 . The prediction of protein membrane topology was performed by TMHMM (Version 2.0) ( https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 ). Alignment of protein structures was done by PyMOL software (Version 2.5). Amino acids sequence alignment was done by ESPript 3.0 ( https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ). To phylogenetically compare StlP with other SPFH proteins, MEGA 7 was used. Amino acid sequences of all SPFH proteins were downloaded from the UniPort database. For phylogenetic analysis of the distribution of StlP, the amino acid sequence of StlP was used to run Position-Specific Iterative (PSI)-BLAST to find homologs in the dataset of 15405 RefSeq representative bacteria and archaea. The homologs with a bitscore > 130 were chosen and subsequently each hit was subjected to membrane topology prediction using TMHMM server (Version 2.0). Only hits with an identical membrane topology were considered valid StlP homologs. The phylogenetic tree was annotated using iTOL ( https://itol.embl.de/ ). Statistical analysis For statistical analyses, GraphPad Prism software (version 8.0.2) was used. 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University","correspondingAuthor":false,"prefix":"","firstName":"Erik","middleName":"","lastName":"Vijgenboom","suffix":""},{"id":266742400,"identity":"bf93608d-783c-4614-84ae-27d50f9983a3","order_by":8,"name":"Gilles van Wezel","email":"","orcid":"https://orcid.org/0000-0003-0341-1561","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Gilles","middleName":"van","lastName":"Wezel","suffix":""},{"id":266742401,"identity":"e40faaa7-0c3f-4a01-89ae-0f141ce89f7c","order_by":9,"name":"Victor Carrion Brava","email":"","orcid":"","institution":"Universidad de Málaga","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"Carrion","lastName":"Brava","suffix":""},{"id":266742402,"identity":"2e356f2a-2887-4122-9bb8-c866e2afb174","order_by":10,"name":"Ariane Briegel","email":"","orcid":"https://orcid.org/0000-0003-3733-3725","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Ariane","middleName":"","lastName":"Briegel","suffix":""},{"id":266742403,"identity":"56d98669-04e1-408b-8b3e-65ef895df50e","order_by":11,"name":"Marc Bramkamp","email":"","orcid":"https://orcid.org/0000-0002-7704-3266","institution":"Kiel University","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Bramkamp","suffix":""}],"badges":[],"createdAt":"2023-12-27 10:02:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3811693/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3811693/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58093-x","type":"published","date":"2025-03-18T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49940120,"identity":"a4194763-2281-49e9-b63e-ee4e9d40f6ca","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":950696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe stomatin-like protein StlP is important for morphogenesis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. coelicolor\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A) \u003c/strong\u003eGrowth of \u003cem\u003eS. coelicolor\u003c/em\u003e M145, its \u003cem\u003estlP \u003c/em\u003emutant and\u003cem\u003e \u003c/em\u003ethe complemented strain (∆\u003cem\u003estlP\u003c/em\u003e+pXZ15) measured using a BioLector. Strains were grown in LPB medium, and biomass was measured in arbitrary unites in triplicates. \u003cstrong\u003e(B) \u003c/strong\u003eColonies of \u003cem\u003eS. coelicolor\u003c/em\u003e M145, its \u003cem\u003estlP \u003c/em\u003emutant and\u003cem\u003e \u003c/em\u003ethe complemented strain (∆\u003cem\u003estlP\u003c/em\u003e+pXZ15) after 5 days of growth.\u003cstrong\u003e (C) \u003c/strong\u003eQuantitative measurement of average colony diameters. Error bars represent the standard error of the mean (***, p\u0026lt;0.0001). \u003cstrong\u003e(D) \u003c/strong\u003eMycelial morphology of the strains grown on LPMA medium. 16 h-old mycelium was labeled with SYTO-9 (stains nucleic acids) and FM5-95 (lipid stain). CWD cells are indicated with the white arrowhead. \u003cstrong\u003e(E) \u003c/strong\u003eStills of Supplementary Movie 1 showing growth of the \u003cem\u003estlP\u003c/em\u003emutant on LPMA medium. Micrographs were taken every 10 min.\u003cstrong\u003e (F) \u003c/strong\u003eThe absence of StlP causes hyperbranching in \u003cem\u003eS. coelicolor\u003c/em\u003e. Hyphae were imaged after 16 h of growth on cellophane membranes overlaying LPMA plates. Note that reintroduction of \u003cem\u003estlP \u003c/em\u003eexpressed from the constitutive \u003cem\u003egapAp \u003c/em\u003epromoter (pXZ15) restores normal branching in the \u003cem\u003estlP \u003c/em\u003emutant. \u003cstrong\u003e(G) \u003c/strong\u003eHistograms showing the tip-to-branch distances in hyphae of M145, its \u003cem\u003estlP \u003c/em\u003emutant\u003cem\u003e \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ethe complemented mutant (∆\u003cem\u003estlP\u003c/em\u003e+pXZ15). Strain were grown for 16 h. For the measurements, \u003cem\u003en\u003c/em\u003e was 188 for M145, 282 for the \u003cem\u003estlP \u003c/em\u003emutant and 192 (complemented mutant). Scale bars represent 5 mm (B), 50 µm (D) and 5 µm (E and F), respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/62ce977832740352962f0315.png"},{"id":49940122,"identity":"14a00ab1-1466-47ac-9e62-9a421b3c41c2","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":960047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStlP localizes to hyphal tips and affects cell wall synthesis. (A) \u003c/strong\u003eLocalization of mCherry-StlP in the wild-type strain and the ∆\u003cem\u003estlP\u003c/em\u003e mutant, both of which carry plasmid pXZ16.\u003cstrong\u003e (B) \u003c/strong\u003eLocalization of DivIVA-mCherry in the wild-type strain and the ∆\u003cem\u003estlP\u003c/em\u003e mutant, both of which carry plasmid pXZ17. Strains were grown in LPB medium for 16 h prior to imaging. The arrowheads indicate the location of DivIVA-mCherry. \u003cstrong\u003e(C) \u003c/strong\u003eVisualization of nascent peptidoglycan in \u003cem\u003eS. coelicolor\u003c/em\u003e strains using fluorescent vancomycin (VanFL) staining. White arrows indicate PG synthesis sites. \u003cstrong\u003e(D) \u003c/strong\u003eDeletion of \u003cem\u003estlP\u003c/em\u003e increases hyphal diameter. Quantification was done by measuring the hyphal diameter after staining with FM5-95. \u003cstrong\u003e(E) \u003c/strong\u003eThe absence of StlP affects proper deposition of the cellulose-like glycan. Foci are either splitting at hyphal tips or diffused along the filament in the ∆\u003cem\u003estlP\u003c/em\u003e mutant. Strains were grown in LPB medium and fluorescent images were taken after 16 h of growth. Bars represent 20 μm. \u003cstrong\u003e(F) \u003c/strong\u003eQuantification of the cellulose-like glycan at hyphal tips using calcofluor white staining. For each tip, the total fluorescence in a square (1.5 μm by 1.5 μm) at the hyphal tip was measured using ImageJ software.\u003cstrong\u003e (G) \u003c/strong\u003eThe absence of the cellulose-like glycan at hyphal tips increases lysozyme sensitivity of the ∆\u003cem\u003estlP\u003c/em\u003e mutant. For each strain, ~1000 spores were plated on nutrient agar plates, and colony numbers were counted after 3 days. The percentage (number of colonies from plates with 0.25 mg ml\u003csup\u003e-1\u003c/sup\u003e lysozyme divided by the number of colonies from plates without lysozyme) was used to evaluate the sensitivity of strains for lysozyme.\u003cstrong\u003e (H) \u003c/strong\u003eCryo-ETs of sacculi of the wild-type and ∆\u003cem\u003estlP\u003c/em\u003e mutant strain (top panel). The bottom panels indicate straightened apical regions of sacculi for the correspnding strain. White arrows indicate contractile injection systems (see Fig. S8 for more examples).\u003cstrong\u003e (I) \u003c/strong\u003eThickness measurement of sacculi of the wild-type and ∆\u003cem\u003estlP\u003c/em\u003e mutant strain. Error bars represent the standard error of the mean (***, P\u0026lt;0.001; ns, non-significance). Bars represent 20 μm (A), 5 μm (B), 1 μm (C),\u0026nbsp; 10 μm (E), 200 nm (H, top panel), and 50 nm (H, bottom panel), respectively. Error bars represent the standard error of the mean (****,P\u0026lt;0.0001; ***, P\u0026lt;0.001; **, P\u0026lt;0.01; ns, not significant).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/b110085370eaa0705667ab9a.png"},{"id":49940123,"identity":"43073ec4-a95c-4a7c-b299-4d1e0ad278af","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1042225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStlP controls membrane fluidity at hyphal tips. (A) StlP assembles into oligomers, consistent with other stomatin proteins. \u003c/strong\u003eThe stomatin domain of StlP (StlP\u003csub\u003eSD\u003c/sub\u003e) was purified and 1 mg ml\u003csup\u003e-1\u003c/sup\u003e of protein was cross-linked with glutaraldehyde (0%, 0.02%, 0.05% and 0.1%) for 10 or 60 min at room temperature. Lane BC and AC represent the protein before and after concentration, respectively. The ladder-like pattern on SDS-PAGE indicates oligomerization.\u003cstrong\u003e (B) \u003c/strong\u003eStlP decamer assembles into a 10-fold symmetric ring structure predicted using AlphaFold. The prediction utilized the amino acid sequence spanning two N-terminal transmembrane helices (TMHs) at positions 106-126 and 149-168, as well as the stomatin domain at positions 209-311 of StlP.\u003cstrong\u003e (C) \u003c/strong\u003eVisualization of fluid region at hyphal tips using DilC\u003csub\u003e12\u003c/sub\u003e staining, in which the 16h-old culture of \u003cem\u003eS. coelicolor\u003c/em\u003e M145, ∆\u003cem\u003estlP \u003c/em\u003emutant and the complemented mutant strain (∆\u003cem\u003estlP\u003c/em\u003e+pXZ15) were incubated with 100 μM DilC\u003csub\u003e12\u003c/sub\u003e and grown for further 3 hours in LPB liquid medium prior to imaging. White arrows indicate fluid regions at hyphal tips. Please note that the extruded CWD cells are highly fluid.\u003cstrong\u003e \u003c/strong\u003eQualitative \u003cstrong\u003e(D)\u003c/strong\u003e and quantitative \u003cstrong\u003e(E)\u003c/strong\u003e analysis of membrane fluidity using Laurdan staining. Mycelia from 16h-old cultures of different strains were collected and labeled with 1mM 100 μM Laurdan\u003cstrong\u003e. \u003c/strong\u003eThe values in the graph \u003cstrong\u003e(E)\u003c/strong\u003e indicate the generalized polarization (GP), which ranges from -1 (more fluid) to +1 (less fluid).\u003cstrong\u003e (F) \u003c/strong\u003eIncreased growth temperature restores membrane fluidity of the ∆\u003cem\u003estlP \u003c/em\u003emutant. Here, strains were grown in LPB liquid medium at 22, 30 or 37⁰C for 16 h and the membrane fluidity is indicated as GP value. Approximately 45 hyphae were measured for each strain. Scale bar represents 5 μm (C), 10 μm (D, main images) and 0.5 μm (D, inlays), respectively. Error bars represent the standard error of the mean (***, P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/561fb5451c45410e939a6851.png"},{"id":49940121,"identity":"d3cd3fd4-148c-4528-856f-212290afc5dc","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of the distribution of StlP proteins in Proteobacteria. \u003c/strong\u003eMaximum-likelihood was used to construct the phylogenetic trees. PSI-BLAST and TMHMM prediction were used to identify StlP homologs in the dataset of 15,405 RefSeq representative bacteria and archaea (left panel). The right panel indicates the distribution of StlP homologs in the genus of actinobacteria. The inner strips indicates different phyla (left panel) or genera (right panel), represented by different colors, while the outside black strip indicates StlP homologs. The green and pink arrowheads represent \u003cem\u003eStreptomyces coelicolor \u003c/em\u003eA3(2) and \u003cem\u003eKitasatospora viridifaciens \u003c/em\u003eDSM40239, respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/67dc75366befa332e92a6058.png"},{"id":49940126,"identity":"af8f736c-3c30-41b1-bf50-f190c45fd42b","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":913509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced robustness of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. viridifaciens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egrowth under hyperosmotic stress conditions through constitutive StlP expression\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A) \u003c/strong\u003eVisualization of fluid membrane regions at hyphal tips of a \u003cem\u003eK. viridifaciens\u003c/em\u003e derivative constitutively expressing StlP (\u003cem\u003eK. viridifaciens+\u003c/em\u003epXZ15) compared to the parental wild-type. Images were obtained after DilC\u003csub\u003e12\u003c/sub\u003e staining of 16h-old cultures followed by growth for 3 hours in LPB liquid medium. White arrows indicate fluid regions at hyphal tips.\u003cstrong\u003e \u003c/strong\u003eQualitative \u003cstrong\u003e(B) \u003c/strong\u003eand quantitative\u003cstrong\u003e (C) \u003c/strong\u003eanalysis of membrane fluidity using Laurdan staining. Mycelia from 16h-old cultures were collected and labeled with 1mM Laurdan\u003cstrong\u003e. \u003c/strong\u003eThe values in the graph \u003cstrong\u003e(C)\u003c/strong\u003e indicate the generalized polarization (GP), which ranges from -1 (more fluid) to +1 (less fluid). \u003cstrong\u003e(D) \u003c/strong\u003eConstitutive expression of \u003cem\u003estlP\u003c/em\u003e from the \u003cem\u003egapAp\u003c/em\u003e promoter increases the average colony size of \u003cem\u003eK. viridifaciens\u003c/em\u003e. Colonies of the wild-type strain and a derivative constitutively expressing StlP (\u003cem\u003eK. viridifaciens+\u003c/em\u003epXZ15) are shown after 7 days of growth.\u003cstrong\u003e (E) \u003c/strong\u003eQuantitative assessment of the average colony diameter.\u003cstrong\u003e (F) \u003c/strong\u003eConstitutive expression of \u003cem\u003estlP\u003c/em\u003e from the \u003cem\u003egapAp\u003c/em\u003e promoter prevents hyperbranching. Individual hyphae were imaged after 16 h of growth on cellophane membranes overlaying LPMA plates.\u003cstrong\u003e (G) \u003c/strong\u003eHistograms indicating the distribution of tip-to-branch distances of \u003cem\u003eK. viridifaciens \u003c/em\u003ewith or without pXZ15. The number of filaments measured for each strain were 219 (\u003cem\u003eK. viridifaciens\u003c/em\u003e) and 189 (\u003cem\u003eK. viridifaciens+\u003c/em\u003epXZ15).\u003cstrong\u003e (H) \u003c/strong\u003eConstitutive expression of StlP (from plasmid pXZ15) blocks the extrusion of wall-deficient cells in \u003cem\u003eK. viridifaciens.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(I)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eQuantification of cell wall-deficient cells was assessed after growing strains in LPB medium for 40 h. Scale bars represent 5 μm (A and F), 10 μm (B, main images), 0.5 μm (B, inlays), 5 mm (D) and 20 μm (H), respectively.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/014612d7a5eb64dbcb91c853.png"},{"id":49940127,"identity":"6851e49e-c89c-43ab-92e0-c997fcbfd9c3","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model for tip growth under conditions of hyperosmotic stress. \u003c/strong\u003eStlP, a stomatin-like protein, undergoes oligomerization, creating an assembly on the membrane that facilitates the formation of microdomains. These microdomains induce local membrane fluidization and serve as a platform for coordinated cell wall synthesis, ensuring normal polar growth. In the absence of StlP (right panel), membrane fluidity diminishes in the tip region, coinciding with the diffusion of apical cellulose-like glycan and peptidoglycan synthesis foci, resulting in the loss of spatially confined cell wall synthesis. This weakens the cell wall, leading to the extrusion of cells with deficient walls. The black arrowhead points to a CWD cell that rebuilds its cell wall.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/2487432fa64f42f268d2df20.png"},{"id":78803150,"identity":"dd161954-5175-4e7b-9662-c9f7b05ffb3e","added_by":"auto","created_at":"2025-03-19 07:09:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6596201,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/51e84e38-11fe-46a6-93d2-573b8e16ec30.pdf"},{"id":49940129,"identity":"530ef5cd-5e7f-4097-afaf-c315c38b4174","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3913512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/7f977a960d6056b38dcfc7b8.pdf"},{"id":49940279,"identity":"caa5ddc7-4568-4198-a40b-810ee36ef093","added_by":"auto","created_at":"2024-01-22 01:44:41","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":556846,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie S1\u003c/p\u003e","description":"","filename":"SupplementaryMovie1.avi","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/c14cef1e692d9efedd2ebe01.avi"},{"id":49940125,"identity":"b4bed4d6-5f5e-441a-9efb-d70612615f5a","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":552486,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie S2\u003c/p\u003e","description":"","filename":"SupplementaryMovie2.avi","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/60e759d60a76c82fc041f0ec.avi"},{"id":49940130,"identity":"6c00b495-97cd-4cd2-9270-61d29d1fe522","added_by":"auto","created_at":"2024-01-22 01:36:42","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":61279596,"visible":true,"origin":"","legend":"Supplementary Movie S3","description":"","filename":"SupplementaryMovie3.avi","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/6e2d5e8ac1dff148bf05cb3f.avi"},{"id":49940128,"identity":"e9c42be4-fd1c-4e44-92b4-3e0523aa4632","added_by":"auto","created_at":"2024-01-22 01:36:41","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16219510,"visible":true,"origin":"","legend":"Supplementary Movie S4","description":"","filename":"SupplementaryMovie4.avi","url":"https://assets-eu.researchsquare.com/files/rs-3811693/v1/ca9237b64044b2974b079654.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The stomatin-like protein StlP organizes membrane microdomains to govern polar growth in filamentous actinobacteria under hyperosmotic stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cell wall is considered an essential structure in bacteria that protects cells from environmental stresses\u003csup\u003e1,2\u003c/sup\u003e. To enable bacterial growth, the cell wall needs to be expanded, which involves inserting new cell wall material at the sites of growth. Elongation of rod-shaped cells typically occurs in two distinct manners\u003csup\u003e3\u003c/sup\u003e. Some rod-shaped bacteria, such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e elongate by incorporating new cell wall material in a rather diffuse manner in the cylindrical part of the cell\u003csup\u003e4\u003c/sup\u003e. By contrast, other bacteria grow by inserting new cell wall material at the cell poles, referred to as polar growth, which is widespread in actinobacteria\u003csup\u003e5,6\u003c/sup\u003e. This mode-of-growth has been well studied in actinomycetes, which are filamentous bacteria that form branched mycelial networks in soil environments. In their natural environment, actinomycetes are often confronted with suboptimal conditions, such as fluctuations in water availability causing dramatic osmotic imbalances\u003csup\u003e7\u003c/sup\u003e. Paradoxically, we recently showed how conditions of hyperosmotic stress causes shedding of the cell wall in several actinomycetes, implying that such conditions interfere with the process of cell wall growth\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePolar growth in actinomycetes is guided by the cytoskeletal protein DivlVA, which localizes in actively growing tips acting as a scaffold for other proteins involved in organizing tip growth, such as the coiled-coil protein Scy and the intermediate filament-like protein FilP\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. Unlike Scy and FilP, DivIVA plays an essential function in polar growth, by directly interacting with the machinery involved in synthesis of peptidoglycan, a major constituent of the cell wall\u003csup\u003e12,13\u003c/sup\u003e. The partial depletion of DivIVA caused hyphal bulging and irregular branching\u003csup\u003e9\u003c/sup\u003e. Scy is an unusual long coiled-coil protein that co-localizes with DivlVA at hyphal tips. Scy was suggested to form higher order assemblies and thereby serves as a hub to stabilize the tip-organizing center, which also includes cell division proteins and proteins involved in chromosome segregation\u003csup\u003e11,14,15\u003c/sup\u003e. Collectively, these proteins make sure that cell wall synthesis is coordinated with chromosome segregation to ensure proper growth and development of the mycelium.\u003c/p\u003e \u003cp\u003eDivlVA also interacts with the putative cellulose synthase CslA\u003csup\u003e16\u003c/sup\u003e. The glycan produced by CslA is thought to provide protection to the tips, which are constantly remodeled during growth, in particular under conditions of osmotic stress\u003csup\u003e16\u003c/sup\u003e. Synthesis of the cellulose-like glycan not only depends on the synthase CslA, but also on a range of other proteins that are all encoded in a conserved gene cluster (Supplementary Fig.\u0026nbsp;1A). Together with CslA, the radical copper oxidase GlxA are the key proteins responsible for synthesis and likely modification of the glycan\u003csup\u003e17,18\u003c/sup\u003e. Following synthesis of the glycan, the lytic polysaccharides monooxygenase LpmP and endoglucanase CslZ facilitate deposition of the glycan chain at the cell surface, possibly by creating a passage through the thick peptidoglycan (PG) layer\u003csup\u003e19\u003c/sup\u003e. The cooperation of CslA/GlxA and CslZ/LpmP implies that a multicomplex is established at the tip related to the proper synthesis and secretion of the cellulose-like glycan.\u003c/p\u003e \u003cp\u003eOne of the proteins in the cellulose biosynthetic gene cluster that has not been studied encodes a stomatin/prohibitin/flotillin/HflK/C (SPFH)-domain protein, hereinafter referred to as StlP (for stomatin-like protein). The SPFH-superfamily proteins facilitate formation of lipid rafts, which in eukaryotes often contain protein complexes that collectively carry out important biological processes, such as ion channel regulation and touch sensation\u003csup\u003e20,21\u003c/sup\u003e. Notably, prokaryotic SPFH proteins, such as flotillins, also form comparable structures known as functional membrane microdomains (FMMs)\u003csup\u003e22\u003c/sup\u003e. These FMMs were shown to be involved in cell wall biosynthesis in \u003cem\u003eB. subtilis\u003c/em\u003e\u003csup\u003e23\u003c/sup\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e. Additionally, it was revealed that, in \u003cem\u003eB. subtilis\u003c/em\u003e, flotillins play a direct role in controlling membrane fluidity homeostasis\u003csup\u003e23\u003c/sup\u003e, with their expression intriguingly regulated by stress-specific signals\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we reveal the pivotal role of StlP in coordinating the creation of a microdomain crucial for sustaining tip growth in \u003cem\u003eStreptomyces coelicolor\u003c/em\u003e during hyperosmotic stress conditions. The absence of StlP leads to anomalous hyphal shape alterations and, notably, the expulsion of cells lacking their cell wall. StlP undergoes polymerization into oligomers and localizes within the membrane, culminating in the formation of a membrane region characterized by heightened fluidity. This phenomenon is essential for harmonizing the growth of both membrane and cell wall during tip extension. Significantly, the ectopic expression of StlP in actinomycetes, known for naturally extruding wall-deficient cells, effectively prevents such extrusion. These findings collectively underscore the significance of StlP in establishing a membrane microdomain at hyphal tips, thereby playing a critical role in facilitating proper cell wall assembly under hyperosmotic stress conditions in filamentous actinobacteria.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eStlP is a stomatin-like protein in\u003c/b\u003e \u003cb\u003eStreptomyces coelicolor\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStlP is encoded in the conserved cellulose biosynthesis gene cluster in streptomycetes and located downstream of \u003cem\u003elpmP\u003c/em\u003e\u003csup\u003e19\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;1A). Analysis of protein domains with InterPro and structure prediction via AlphaFold reveal that StlP contains three domains: a disordered region (aa 1-100), followed by a transmembrane hairpin composed by two helices (aa 106\u0026ndash;126 and 149\u0026ndash;168), and a SPFH (stomatin, prohibitin, flotillin, HflK/C) domain (aa 209\u0026ndash;311) (Supplementary Fig.\u0026nbsp;1B, Supplementary Fig.\u0026nbsp;1C).\u003c/p\u003e \u003cp\u003eConsidering the diverse membrane topologies observed in members of the SFPH superfamily\u003csup\u003e26\u003c/sup\u003e, we examined the membrane topology of StlP to determine its classification within the family of SPFH-containing proteins. \u003cem\u003eIn silico\u003c/em\u003e analyses predicted that the C-terminal SPFH domain of StlP is in the cytoplasm (Supplementary Fig.\u0026nbsp;1D). To validate this prediction, we employed a β-lactamase assay in which this enzyme was fused to the C-terminus of StlP as described\u003csup\u003e27\u003c/sup\u003e. \u003cem\u003eE. coli\u003c/em\u003e cells expressing StlP-BlaM\u003csub\u003eNS\u003c/sub\u003e fusions demonstrate sensitivity to ampicillin, in contrast to cells expressing BlaM\u003csub\u003eFL\u003c/sub\u003e with its original signal peptide (Supplementary Fig.\u0026nbsp;1E). This result confirms that the C-terminal SPFH domain of StlP is situated in the cytoplasm, consistent with the observed membrane topology in podocin/stomatin family proteins\u003csup\u003e20\u003c/sup\u003e and its N-terminal transmembrane hairpin. In contrast to StlP, other SPFH-like proteins from \u003cem\u003eS. coelicolor\u003c/em\u003e have a significantly different predicted membrane topology (Supplementary Fig.\u0026nbsp;2). A phylogenetic analysis, utilizing amino acid sequences from well-known prokaryotic and eukaryotic SPFH proteins, revealed that \u003cem\u003eS. coelicolor\u003c/em\u003e StlP and Mouse stomatin STOML-1 form a monophyletic clade, indicating that StlP resembles a stomatin-like protein (Supplementary Fig.\u0026nbsp;3A). Further sequence alignment of StlP with other stomatins indicates that \u003cem\u003eStreptomyces\u003c/em\u003e StlP contains the signature proline\u003csup\u003e28\u003c/sup\u003e residue required for membrane hairpin formation and a comparable domain arrangement (Supplementary Fig.\u0026nbsp;3B). This alignment suggests that StlP shares more similarities with eukaryotic stomatins than prokaryotic stomatins. In summary, these findings collectively establish StlP as a stomatin-like protein within the prokaryotic family of SPFH proteins.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStlP is important for morphogenesis under osmotic stress conditions\u003c/h2\u003e \u003cp\u003eTo investigate the function of StlP, a \u003cem\u003estlP\u003c/em\u003e mutant was created that contained a Tn5062 transposon insertion, positioned upstream of the stomatin domain. This constructed \u003cem\u003estlP\u003c/em\u003e mutant was used to study its phenotype in various conditions. The \u003cem\u003estlP\u003c/em\u003e mutant displayed significantly reduced growth rates in both TSBS and LPB liquid medium, which contain 10 and 22% sucrose, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;4A). On MS agar medium (without sucrose), no apparent differences in growth were observed between the parent and the \u003cem\u003estlP\u003c/em\u003e mutant (Supplementary Fig.\u0026nbsp;4B). By contrast, when grown on LPMA agar medium (containing 22% sucrose), the average diameter of individual colonies of the \u003cem\u003estlP\u003c/em\u003e mutant (1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm) was significantly reduced compared to the wild-type strain (3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that growth was severely hampered. Furthermore, we noticed that excess membrane was extruded from hyphal tips of the \u003cem\u003estlP\u003c/em\u003e mutant, as evident in the FM5-95 panels comparing M145 and ∆\u003cem\u003estlP\u003c/em\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. Additionally, we observed many DNA-containing vesicles present in the medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) that were reminiscent of cell wall-deficient cells extruded by several filamentous actinobacteria. These vesicles were absent in the parental strain, consistent with earlier findings\u003csup\u003e8\u003c/sup\u003e. Time-lapse imaging revealed that the vesicles were extruded from hyphal tips (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Supplementary Movies 2). Quantification revealed that the mutant formed 2.5x10\u003csup\u003e5\u003c/sup\u003e vesicles ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while none were found in the parental strain (Supplementary Fig.\u0026nbsp;5). To confirm that these phenotypes were caused by the absence of StlP, we introduced plasmid pXZ15 in the mutant, in which \u003cem\u003estlP\u003c/em\u003e is expressed from the constitutive \u003cem\u003egapAp\u003c/em\u003e promoter. Reintroduction of this plasmid partially restored the growth speed in liquid media (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4A) and the average colony diameter on LPMA medium (2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), while extrusion of membranes and DNA-containing vesicles was also reduced by 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eMicroscopy analysis also indicated that the hyphal branching pattern was affected by the deletion of \u003cem\u003estlP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Supplementary Fig.\u0026nbsp;4D, Supplementary Movies 1 and 2). More specifically, the number of branches was dramatically increased in the \u003cem\u003estlP\u003c/em\u003e mutant when grown under hyperosmotic stress conditions (LPMA medium) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). To quantitatively compare branching between the strains, we measured the distance from the tip to the proximal branching point in hyphae (Supplementary Fig.\u0026nbsp;4D). Deletion of \u003cem\u003estlP\u003c/em\u003e changed the distribution of the tip-to-branch distances significantly. We found that more than 60% of all hyphae in ∆\u003cem\u003estlP\u003c/em\u003e had a proximal branch within the first 5 \u0026micro;m from the tip (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), as compared to approximately 15% for the parent and the complemented mutant. Also, no hyphae of the \u003cem\u003estlP\u003c/em\u003e mutant branched further than 35 \u0026micro;m from the tip. Additionally, when grown on MS medium, hyphae with a tip-to-branch distance less than 10 \u0026micro;m accounted for 3.6% and 8.9% of the parent and mutant strain, respectively (Supplementary Fig.\u0026nbsp;4E). This suggests a mild impact on the branching pattern due to the deletion of \u003cem\u003estlP\u003c/em\u003e under normal growth conditions. Taken together, these findings underscore that the absence of \u003cem\u003estlP\u003c/em\u003e significantly influences the growth and morphogenesis of \u003cem\u003eS. coelicolor\u003c/em\u003e, particularly manifesting under conditions of hyperosmotic stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStlP is important for spatially confining cell wall synthesis to hyphal tips\u003c/h2\u003e \u003cp\u003eTo investigate the localization of StlP, we introduced pXZ16 into \u003cem\u003eS. coelicolor\u003c/em\u003e M145, thereby constitutively expressing a C-terminal mCherry fusion to StlP. Foci of StlP-mCherry mostly localized at growing tips and emerging branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We also localized the polar growth determinant DivIVA in the presence and absence of StlP by expressing a DivIVA-mCherry fusion from the constitutive \u003cem\u003egapAp\u003c/em\u003e promoter. Interestingly, in the absence of StlP, DivIVA-mCherry was not only localized to hyphal tips but was also found in numerous foci along the cylindrical part of the filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting the cell wall synthesis was no longer confined to the apex. In agreement, nascent PG was incorporated at multiple sites along the filament in the \u003cem\u003estlP\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), coinciding with an increase in the average diameter of the hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, we noticed that multiple synthesis foci of the cellulose-like glycan by CslA appeared at established and emerging hyphal tips, which is contrary to the parental strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Deposition of the cellulose-like glycan was affected in the \u003cem\u003estlP\u003c/em\u003e mutant and was no longer spatially confined to the hyphal tip (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Quantitative analysis indicated that the absence of StlP let to some 60% reduced accumulation of glycans at hyphal tips, indicated by calcofluor white staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Furthermore, the reduced glycan levels made the \u003cem\u003estlP\u003c/em\u003e mutant sensitive to lysozyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), which was previously observed in other mutants affected in apical glycan deposition. Glycan deposition and lysozyme resistance were restored when the complementation plasmid pXZ15 was introduced in the \u003cem\u003estlP\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo accurately evaluate the effect of the absence StlP on cell wall thickness of \u003cem\u003eS. coelicolor\u003c/em\u003e, we conducted a thorough examination by preparing and imaging sacculi of M145 and the ∆\u003cem\u003estlP\u003c/em\u003e strains using cryo-electron tomography (cryo-ET). The analysis of cell wall thickness measurement revealed a significant reduction in the overall cell wall thickness upon \u003cem\u003estlP\u003c/em\u003e deletion compared to the parental strain. Specifically, the thickness in the apical region decreased from 35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 nm to 6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nm, while the thickness in the subapical region was reduced from 37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 nm to 9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). These results demonstrate the importance of StlP for cell wall synthesis and thickness of \u003cem\u003eS. coelicolor\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo establish how StlP contributes to delocalized cell wall synthesis, we tested interactions of StlP with the proteins involved in tip growth and cellulose biosynthesis. To this end, constructs were generated that produced C-terminal fusions of StlP, LpmP, SCO2835, CslA, GlxA, CslZ, DivIVA, Scy and FilP to either the T25 or T18 fragments of the adenylate cyclase, respectively. Co-transformation of these constructs in \u003cem\u003eE. coli\u003c/em\u003e BTH101 revealed that StlP robustly interacts with LpmP, SCO2835, CslA and CslZ, but also with itself and weakly interacts with GlxA (Supplementary Fig.\u0026nbsp;6). Furthermore, StlP did not interact with DivIVA, Scy or FilP (Supplementary Fig.\u0026nbsp;6). Thus, StlP directly interacts with components of the cellulose biosynthesis complex and spatially confines cell wall synthesis to hyphal tips in \u003cem\u003eS. coelicolor\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of\u003c/b\u003e \u003cb\u003estlP\u003c/b\u003e \u003cb\u003einduces cell death in\u003c/b\u003e \u003cb\u003eS. coelicolor\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe noticed a significant abundance of contractile injection system (CIS) structures within the sacculi of the \u003cem\u003estlP\u003c/em\u003e mutant cultivated for 16 h, in contrast to the absence of these structures in the sacculi of an equivalently aged parental strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These CIS structures were recently found to be involved in programmed cell death regulation\u003csup\u003e29,30\u003c/sup\u003e. The observation of CIS led us to investigate if the deletion of \u003cem\u003estlP\u003c/em\u003e increased cell death of \u003cem\u003eS. coelicolor\u003c/em\u003e. To investigate this, M145 and its \u003cem\u003estlP\u003c/em\u003e mutant were grown for 16 h on LPMA medium. Cell death in the mycelia was measured using a bacterial viability assay\u003csup\u003e31\u003c/sup\u003e (see Materials and Methods). In principle, when SYTO9 and propidium iodide (PI) nucleic acid stains are present simultaneously, stained viable mycelia and dead mycelia exhibited green, and red fluorescence, respectively. The live/dead (SYTO9/PI) ratio was notably reduced in the \u003cem\u003estlP\u003c/em\u003e mutant when compared to the parent strain (see Supplementary Fig.\u0026nbsp;8). This decrease suggests increased cell death in the \u003cem\u003estlP\u003c/em\u003e mutant, aligning with our findings that the deletion of \u003cem\u003estlP\u003c/em\u003e significantly impeded the growth of liquid-cultured \u003cem\u003eS. coelicolor\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, supplementary Fig.\u0026nbsp;4A) Altogether, these results imply that deletion of \u003cem\u003estlP\u003c/em\u003e contributes to increased cell death of \u003cem\u003eS. coelicolor\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStlP oligomerizes and forms membrane microdomain at hyphal tips of\u003c/b\u003e \u003cb\u003eS. coelicolor\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacterial two-hybrid analysis suggested that StlP interacts with itself but also with components of the machinery involved in glycan synthesis (Supplementary Fig.\u0026nbsp;6). To verify if StlP interacts with itself via the stomatin domains, the corresponding domain (aa 204\u0026ndash;326; referred to as StlP\u003csub\u003eSD\u003c/sub\u003e) was expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). The StlP\u003csub\u003eSD\u003c/sub\u003e monomer was expressed, showing a predicted molecular mass of 18 kDa (lane BC, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). When samples were concentrated to 1 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a ladder of oligomers was found (lane AC), suggesting that monomers can self-assemble. Notably, less monomers and more crosslinked highly ordered polymers were observed with increasing concentrations of the cross-linker glutaraldehyde (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results suggest that StlP can assemble into oligomers via its stomatin domain at high concentrations, which in the full-length protein would probably result in formation of a membrane microdomain via its two N-terminal transmembrane helices. To corroborate this hypothesis, the partial structure of SltP (aa 106\u0026ndash;326), which covers the two N-terminal TMHs (aa 106\u0026ndash;126 and aa 149\u0026ndash;168) and the stomatin domain (aa 209\u0026ndash;311) was predicted using AlphaFold 2.0. Given that the putative stomatin domain of FliL from the marine bacterium \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, the only structure known of a bacterial stomatin, assembles into a symmetric ring consisting of 10 monomers, 10 StlP monomers were used for prediction of the structure. This predicted that the cytoplasmic stomatin domain of StlP assembles into a highly ordered multimer via an end-to-end interaction pattern, in which the N-terminal transmembrane helical hairpins of the monomers contribute to forming a symmetric ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn bacteria, SPFH proteins are involved in membrane fluidity homeostasis, evidenced by FloT and FloA that fluidize the membrane and absence of them leads to an overall rigidification\u003csup\u003e23,32\u003c/sup\u003e. We therefore predicted that the oligomerization of StlP leads to the formation of a local membrane \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003er\u003c/span\u003eegion with \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ei\u003c/span\u003encreased \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eluidity (RIF) at hyphal tips. To verify this prediction, we stained mycelia of \u003cem\u003eS. coelicolor\u003c/em\u003e with DilC\u003csub\u003e12\u003c/sub\u003e, a lipid dye with high specificity for fluid membranes due to its short hydrocarbon tail\u003csup\u003e33,34\u003c/sup\u003e. We observed bright DilC\u003csub\u003e12\u003c/sub\u003e staining RIFs at hyphal tips, and also along hyphae grown for 16 h in LPB medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The signal corresponding to DilC\u003csub\u003e12\u003c/sub\u003e staining was absent from hyphal tips of the \u003cem\u003estlP\u003c/em\u003e mutant and restored in the complemented strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggest that hyphal tips possess a RIF, which is dependent on the presence of StlP. To substantiate the presence of RIFs at hyphal tips, we conducted a quantitative assessment of membrane fluidity using the membrane-intercalating dye Laurdan. This dye exhibits a shift in fluorescence emission wavelength based on membrane fluidity\u003csup\u003e33,35\u003c/sup\u003e. After culturing mycelia of M145 and its \u003cem\u003estlP\u003c/em\u003e mutant in LPB medium for 16 hours, we stained them with Laurdan and then calculated the general polarization (GP) value at the tip region of each hypha (see Materials and Methods). In the absence of \u003cem\u003estlP\u003c/em\u003e, the average GP value (-0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) increased compared to its parental strain (-0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13), inferring a decreased membrane fluidity in the \u003cem\u003estlP\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Furthermore, the apical membrane fluidity of the \u003cem\u003estlP\u003c/em\u003e mutant could be restored by raising the growth temperature to 37 ⁰C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), which aligns with the idea that elevated temperatures contribute to membrane phase transitions, leading to an increased fluidity\u003csup\u003e36\u003c/sup\u003e. Altogether, these results reveal that StlP oligomerizes and forms membrane domains with enhanced membrane fluidity at hyphal tips of \u003cem\u003eS. coelicolor\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMorphogenesis controlled by StlP is conserved in filamentous actinobacteria\u003c/h3\u003e\n\u003cp\u003eOur results indicate that StlP contributes to proper cell wall synthesis and membrane organization under hyperosmotic stress, and thereby controls morphogenesis of \u003cem\u003eS. coelicolor\u003c/em\u003e. To see how prevalent StlP is, we combined PSI-BLAST and TMHMM prediction to identify stomatin homologues of StlP in the dataset of 15045 RefSeq representative bacteria and archaea. This analysis showed that the majority of StlP homologs are present in actinobacteria, while a few are also present in \u003cem\u003eRhizobium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, inside the filamentous actinobacteria, StlP orthologs are present in genera including \u003cem\u003eStreptomyces, Kitasatospora\u003c/em\u003e and \u003cem\u003eStreptacidiphillus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In some clades of these bacteria, all members have an orthologue of StlP, while in others StlP is less common or virtually absent. Notably, \u003cem\u003eKitasatospora viridifaciens\u003c/em\u003e DSM40239 is among the clades that lack StlP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo see how widespread the function of StlP is in other actinobacteria, the construct pXZ15, wherein the \u003cem\u003estlP\u003c/em\u003e was expressed from the constitutive \u003cem\u003egapAp\u003c/em\u003e promoter, was introduced into \u003cem\u003eKitasatospora viridifaciens\u003c/em\u003e DSM40239 via conjugation, which is known to extrude wall-deficient cells under hyperosmotic stress\u003csup\u003e8,37\u003c/sup\u003e. Importantly, constitutive expression of \u003cem\u003estlP\u003c/em\u003e induced the formation of a fluid membrane microdomain at hyphal tips in \u003cem\u003eK. viridifaciens\u003c/em\u003e, as evidenced by DilC\u003csub\u003e12\u003c/sub\u003e staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, constitutive expression of \u003cem\u003estlP\u003c/em\u003e significantly increased the average membrane fluidity of hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and allowed colonies to cope much better with hyperosmotic stress, as shown by the strongly increased colony diameter of the strain expressing \u003cem\u003estlP\u003c/em\u003e (2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm) as compared to the parental strain (1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mm) and by the reduced lateral branching of \u003cem\u003eK. viridifaciens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, constitutive expression of \u003cem\u003estlP\u003c/em\u003e largely abolished the extrusion of wall-deficient cells in \u003cem\u003eK. viridifaciens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). By contrast, the constitutive expression of the HflK/C-like protein BOQ63_030050 in \u003cem\u003eK. viridifaciens\u003c/em\u003e, which also belongs to the SPFH superfamily of proteins and shares 21% identity with StlP had no effect on the extrusion of wall-deficient cells (Supplementary Fig.\u0026nbsp;9, 10). These results demonstrate that StlP controls growth of filamentous actinobacteria under hyperosmotic stress by the localized control of membrane fluidity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStomatin-like proteins are ubiquitous in all domains of life. A universal feature of these proteins is to form functional nanoscale microdomains in biological membranes. In turn, these microdomains serve as platforms to locate protein complexes involved in important biological processes, such as transducing mechanosensory signals in mice and the modulation of ion channels in mammalian cells\u003csup\u003e38\u0026ndash;40\u003c/sup\u003e. The recent structural analysis of the stomatin-like protein FliL from \u003cem\u003eVibrio alginolyticus\u003c/em\u003e showed that FliL shares some structural elements with eukaryotic stomatins, suggesting that stomatin-like proteins are conserved from mammals to bacteria\u003csup\u003e40\u003c/sup\u003e. Here, we identified the stomatin-like protein StlP as a novel polar growth determinant in filamentous actinobacteria. We show that StlP is crucial for spatially organizing cell wall synthesis at hyphal tips in conditions of hyperosmotic stress, which likely provides competitive benefit to microbes frequently exposed to such conditions.\u003c/p\u003e \u003cp\u003eEvolutionary investigation showed the group of stomatins are ancient and their evolution likely occurred at an early stage in the evolution of prokaryotes\u003csup\u003e41\u003c/sup\u003e. Most prokaryotic stomatins (so-called p-stomatins) have been found in archaeal species, for instance in isolates from environments with high temperatures (\u003cem\u003ePyrococcus horikoshii\u003c/em\u003e, \u003cem\u003eArchaeoglobus fulgidus\u003c/em\u003e and \u003cem\u003eMethanothermobacter thermautotrophicus\u003c/em\u003e) and isolates from sediments near the sea (\u003cem\u003eAeropyrum pernix\u003c/em\u003e)\u003csup\u003e41,42\u003c/sup\u003e. Some p-stomatins exist in bacterial species, such as in \u003cem\u003ePhotobacterium aphoticum\u003c/em\u003e isolated from coastal water\u003csup\u003e43\u003c/sup\u003e and the marine bacterium \u003cem\u003eVibrio alginolyticus\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e. However, their roles have not been characterized well. Excitingly, StlP, the p-stomatin of \u003cem\u003eS. coelicolor\u003c/em\u003e, appears to play a pivotal role in regulating tip growth in conditions of hyperosmotic stress. Interestingly, \u003cem\u003eS. coelicolor\u003c/em\u003e was originally isolated from the beach, which makes it plausible that \u003cem\u003eS. coelicolor\u003c/em\u003e, contrary to \u003cem\u003eK. viridifaciens\u003c/em\u003e isolated from mountain soil, is better adapted to salt-rich environments, for instance by having StlP.\u003c/p\u003e \u003cp\u003eStomatins oligomerize in cell membranes through interactions between their stomatin-domains, thus providing a scaffold for numerous other proteins. This functionality has been demonstrated for several stomatins, including mouse stomatin\u003csup\u003e39\u003c/sup\u003e, \u003cem\u003ePyrococcus horikoshii\u003c/em\u003e stomatin\u003csup\u003e44\u003c/sup\u003e and the stomatin FliL of \u003cem\u003eVibrio alginolyticus\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e. Cross-linking studies and structure predictions demonstrate that the stomatin domain of StlP oligomerizes and that StlP interacts with the machinery involved in synthesis of a cellulose-like glycan. One of these proteins is the cellulose synthase-like protein CslA, which in turn directly interacts with DivIVA\u003csup\u003e16\u003c/sup\u003e. Interestingly, PG synthesis of actinomycetes is directed at hyphal tips in a DivIVA-dependent manner. More specifically, DivIVA recruits penicillin-binding proteins (PBPs) during polarized growth, as deduced from the detected interaction between DivIVA and PBP3 in \u003cem\u003eM. tuberculosis\u003c/em\u003e\u003csup\u003e11,12\u003c/sup\u003e. StlP seems to contribute to establishing a localized region in the cellular membrane carrying crucial components of the so-called tip organizing center, which controls cell wall synthesis during the polar growth in filamentous actinobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Indeed, the observation of diffused PG and surface cellulose synthesis foci and dramatically reduced cell wall thickness of the \u003cem\u003estlP\u003c/em\u003e mutant indicated that PG synthesis is significantly weakened upon the deletion of \u003cem\u003estlP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We thus propose a model for the cell wall synthetic machinery at hyphal tips when filamentous actinobacteria grow in hyperosmotic stress conditions. In this model, DivIVA guides the machinery responsible for peptidoglycan (PG) and surface cellulose-like glycan synthesis to the poles through interactions with CslA and PBPs. At these poles, the systems are restricted by the formation of StlP rings, which, in turn, induce localized membrane fluidization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The synthesis of the surface cellulose-like glycan involves substantial redox reactions facilitated by the presence of LpmP and GlxA. Here, the StlP ring, may serve to restrict oxidative stress within the ring, shielding proteins outside the StlP from this stress. Meanwhile, the StlP ring enables these filamentous actinobacteria to produce robust cell walls during polarized growth under conditions of hyperosmotic stress. Without StlP, the membrane fluidity in the tip region decreases. Combined with the loss of localized cell wall synthesis, this leads to the extrusion of wall-deficient cells. Reversely, constitutive expression of StlP in species that naturally lack this protein prevents the typical extrusion of wall-deficient cells. Taken together, this work provides a plausible mechanism how wall-deficient cells are extruded in polar-growing actinobacteria. By interrupting cell wall synthesis, by exposing the cells to hyperosmotic stress or antibiotics, the coordinated balance between membrane and cell wall synthesis is lost. In turn, this leads to shedding of excess membranes from the polar growth sites and is facilitated by the localized weakening of the cell wall.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, our work for the first time characterizes a stomatin-like protein that regulates tip growth in conditions of hyperosmotic stress. The strong phenotype associated with its absence make this also an interesting candidate to target in pathogenic bacteria that grow from the cell poles. In this context, it is noteworthy that mycobacteria have the ability to adopt a wall-deficient lifestyle\u003csup\u003e45\u003c/sup\u003e, and it\u0026rsquo;s worth highlighting that \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e possesses a StlP homolog.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains used in this study are listed in Supplementary Table\u0026nbsp;1. Solid MS (Mannitol Soy flour) medium\u003csup\u003e46\u003c/sup\u003e was used for collection of \u003cem\u003eStreptomyces\u003c/em\u003e spores and for conjugation experiments, while MYM medium\u003csup\u003e47\u003c/sup\u003e was used for obtaining \u003cem\u003eKitasatospora\u003c/em\u003e spores. To compare colony sizes and observe the release of cell wall-deficient (CWD) cells under hyperosmotic stress, solid LPMA medium\u003csup\u003e8\u003c/sup\u003e was used. TSBS\u003csup\u003e46\u003c/sup\u003e medium was used to grow \u003cem\u003eStreptomyces\u003c/em\u003e in liquid medium without hyperosmotic stress. For quantification of the number of CWD cells, as well as measurement of hyphal diameters and membrane fluidity, liquid L-phase broth (LPB) was used\u003csup\u003e8\u003c/sup\u003e. Briefly, 10\u003csup\u003e6\u003c/sup\u003e CFU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e spores were inoculated in 20 ml LPB in 50 ml flasks without coil while shaking at 100 rpm ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eKitasatospora\u003c/em\u003e strains were grown at 30\u0026deg;C. For hyphal branching detection, spores were inoculated onto cellophane membranes overlaying LPMA plates, which were then incubated at 30\u0026deg;C for 16 h prior to analysis. Lysozyme sensitive assay were performed essentially as described\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha;\u003csup\u003e48\u003c/sup\u003e was used for cloning and \u0026beta;-lactamase experiments. \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)\u003csup\u003e49\u003c/sup\u003e and BTH101\u003csup\u003e50\u003c/sup\u003e were used in protein expression and bacterial two-hybrid assays, respectively. For conjugation, \u003cem\u003eE. coli\u003c/em\u003e ET12567 harboring pUZ8002 was used. All \u003cem\u003eE. coli\u003c/em\u003e strains were grown in LB medium at 37\u0026deg;C with appropriate antibiotics, if needed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids and primers used in this study are listed in Supplementary Table\u0026nbsp;2 and Supplementary Table\u0026nbsp;3, respectively. For complementation of the ∆\u003cem\u003estlP\u003c/em\u003e mutant, the coding sequence of \u003cem\u003estlP\u003c/em\u003e (SCO2834) was amplified from genomic DNA of \u003cem\u003eS. coelicolor\u003c/em\u003e with primers stlP-F/stlP-R1. The PCR product was ligated as an NdeI/BamHI fragment into plasmid hpXZ2\u003csup\u003e19\u003c/sup\u003e harboring the constitutive \u003cem\u003egapAp\u003c/em\u003e prompter of SCO1947, yielding plasmid pXZ15.\u003c/p\u003e\n\u003cp\u003eFor localization of StlP and DivlVA, the mCherry reporter was fused to the C-terminus of these two proteins. Coding sequences for \u003cem\u003estlP\u003c/em\u003e and \u003cem\u003edivlVA\u003c/em\u003e were amplified from genomic DNA of \u003cem\u003eS. coelicolor\u003c/em\u003e with primers stlP-F/stlP-R2 and 2077-F/2077-R, respectively. The gene encoding for mCherry was amplified from plasmid pGWS791\u003csup\u003e27\u003c/sup\u003e using primers mCh-F/mCh-R. After digestions with NdeI/HindIII (for \u003cem\u003estlP\u003c/em\u003e and \u003cem\u003edivlVA\u003c/em\u003e) and HindIII/XbaI (for \u003cem\u003emCherry\u003c/em\u003e), combinations of \u003cem\u003estlP/mCherry\u003c/em\u003e and \u003cem\u003edivlVA/mCherry\u003c/em\u003e were ligated with plasmid hpXZ2 that was cut with NdeI/XbaI, generating plasmid pXZ16 and pXZ17, respectively.\u003c/p\u003e\n\u003cp\u003eTo constitutively express the HflK/C-like protein BOQ63_030050 in \u003cem\u003eKitasatospora viridifaciens\u003c/em\u003e and the \u003cem\u003eS. coelicolor stlP\u003c/em\u003e mutant, the coding sequence of BOQ63_030050 was amplified from genomic DNA of \u003cem\u003eK. viridifaciens\u003c/em\u003e DSM40239 with primers 0050-F/0050-R. The PCR product was ligated as an NdeI/XbaI fragment into plasmid hpXZ2 harboring the constitutive \u003cem\u003egapAp\u003c/em\u003e prompter, yielding plasmid pXZ41.\u003c/p\u003e\n\u003cp\u003eTo express the stomatin-encoding domain of StlP (StlP\u003csub\u003eSD\u003c/sub\u003e) in \u003cem\u003eE. coli\u003c/em\u003e, nucleotides 610\u0026ndash;1107 of \u003cem\u003estlP\u003c/em\u003e were amplified from \u003cem\u003eS. coelicolor\u003c/em\u003e genomic DNA with primers 2834\u003csub\u003esto\u003c/sub\u003e-F/2834\u003csub\u003esto\u003c/sub\u003e-R and ligated into pET28a plasmid as a NdeI and HindIII fragment, yielding plasmid pXZ18. Consequently, StlP\u003csub\u003eSD\u003c/sub\u003e was expressed carrying a N-terminal Histidine-tag (6xHis).\u003c/p\u003e\n\u003cp\u003eAll plasmids were introduced into \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eStreptomyces\u003c/em\u003e via heat-shock transformation\u003csup\u003e51\u003c/sup\u003e and conjugation\u003csup\u003e46\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInactivation of stlP in Streptomyces\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor inactivation of \u003cem\u003estlP\u003c/em\u003e in \u003cem\u003eS. coelicolor and S. lividans\u003c/em\u003e, we used cosmid StE20 carrying the Tn5062 transposon inserted after nucleotide position 892 relative to the start site of \u003cem\u003estlP\u003c/em\u003e (kindly provided by Prof. Paul Dyson, see Supplementary Table 2). This cosmid was introduced into \u003cem\u003eS. coelicolor\u003c/em\u003e via conjugation using ET12567/pUZ8002\u003csup\u003e46\u003c/sup\u003e, after which exconjugants were screened as described\u003csup\u003e52\u003c/sup\u003e. Colonies that were kanamycin-sensitive and apramycin-resistant were selected and used for further analysis. Mutants carrying the expected phenotype were verified by sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth curve generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor preparing germinated spores, spores of different strains were resuspended in double-strength germination medium\u003csup\u003e46\u003c/sup\u003e at a final concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and incubated at 30 ⁰C for 6\u0026thinsp;~\u0026thinsp;8 h while shaking at 200 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For generating the biomass growth curve, the RoboLector L-4-BL-II equipped with a parallelized shaken cultivation device was used\u003csup\u003e53\u003c/sup\u003e. Briefly, the germinated spores were centrifuged and resuspended in either TSBS or LPB media before being distributed (1 ml per well) into a 48-well FlowerPlates (Basesweiler Germany). The temperature and humidity were set at 30 ⁰C and 85%, respectively. The biomass was collected automatically and measured in arbitrary units. All measurements were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial 2-hybrid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess interactions of StlP with other tip-localizing proteins, the bacterial hybrid assay was used\u003csup\u003e50\u003c/sup\u003e. Therefore, \u003cem\u003elpmP\u003c/em\u003e (SCO2833), \u003cem\u003estlP\u003c/em\u003e (SCO2834), \u003cem\u003esco2835\u003c/em\u003e, \u003cem\u003ecslA\u003c/em\u003e (SCO2836), \u003cem\u003eglxA\u003c/em\u003e (SCO2837), \u003cem\u003ecslZ\u003c/em\u003e (SCO2838), \u003cem\u003edivlVA\u003c/em\u003e (SCO2077), \u003cem\u003efilP\u003c/em\u003e (SCO5396) and \u003cem\u003escy\u003c/em\u003e (SCO5397) were amplified using primers Th2833-F/Th2833-R, Th2834-F/Th2834-R, Th2835-F/Th2835-R, Th2836-F/Th2836-R, Th2837-F/Th2837-R, Th2838-F/Th2838-R, divlVA-F/divlVA-R, filP-F/filP-R and scy-F/scy-R, respectively. All amplified DNA fragments were cloned into the pKT25 and pUT18C plasmids using EcoRI and XbaI, yielding plasmids pXZ19 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003elpmP\u003c/em\u003e), pXZ20 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003estlP\u003c/em\u003e), pXZ21 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003estlP\u003c/em\u003e), pXZ22 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003eSCO2835\u003c/em\u003e), pXZ23 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003eSCO2835\u003c/em\u003e), pXZ24 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003ecslA\u003c/em\u003e), pXZ25 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003ecslA\u003c/em\u003e), pXZ26 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003eglxA\u003c/em\u003e), pXZ27 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003eglxA\u003c/em\u003e), pXZ28 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003ecslZ\u003c/em\u003e), pXZ29 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003edivlVA\u003c/em\u003e), pXZ30 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003edivlVA\u003c/em\u003e), pXZ31 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003efilP\u003c/em\u003e), pXZ32 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003efilP\u003c/em\u003e), pXZ33 (pKT25\u0026thinsp;+\u0026thinsp;\u003cem\u003escy\u003c/em\u003e) and pXZ34 (pUT18C\u0026thinsp;+\u0026thinsp;\u003cem\u003escy\u003c/em\u003e) (see Supplementary Table\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e BT101 carrying combinations of these constructs were used in bacterial 2-hybrid experiments to evaluate protein interactions as described\u003csup\u003e54\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTopology determination of StlP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the transmembrane topology of StlP, the \u0026beta;-lactamase\u003cem\u003e-\u003c/em\u003eencoding gene \u003cem\u003eblaM\u003c/em\u003e without its signal sequence was fused to the 3\u0026rsquo; end of \u003cem\u003estlP\u003c/em\u003e, as described previously\u003csup\u003e27\u003c/sup\u003e. Only if BlaM is secreted, cells will be resistant to ampicillin. To this end, a \u003cem\u003eblaM\u003c/em\u003e variant lacking the region encoding the signal sequence for secretion (\u003cem\u003eblaM\u003c/em\u003e\u003csub\u003e\u003cem\u003eNS\u003c/em\u003e\u003c/sub\u003e) was amplified from plasmid pHJL401\u003csup\u003e55\u003c/sup\u003e with primers blaM-F/blaM-R. In parallel, full length \u003cem\u003eblaM\u003c/em\u003e (including the region for the signal sequence, hereinafter referred to as \u003cem\u003eblaM\u003c/em\u003e\u003csub\u003e\u003cem\u003eFL\u003c/em\u003e\u003c/sub\u003e) was amplified from the same plasmid using the blaM\u003csub\u003eFL\u003c/sub\u003e-F/blaM-R primers. The \u003cem\u003estlP\u003c/em\u003e gene was amplified from \u003cem\u003eS. coelicolor\u003c/em\u003e genomic DNA using primers stlP-F/\u003cem\u003estlP\u003c/em\u003e-R3. Subsequently, the amplified products were cut using restriction enzymes NdeI-HindIII (\u003cem\u003estlP\u003c/em\u003e), HindIII-EcoRI (\u003cem\u003eblaM\u003c/em\u003e\u003csub\u003e\u003cem\u003eNS\u003c/em\u003e\u003c/sub\u003e) and NdeI-EcoRI (\u003cem\u003eblaM\u003c/em\u003e\u003csub\u003eFL\u003c/sub\u003e), and digested combinations of \u003cem\u003estlP\u003c/em\u003e/\u003cem\u003eblaM\u003c/em\u003e\u003csub\u003e\u003cem\u003eNS\u003c/em\u003e\u003c/sub\u003e, and \u003cem\u003eblaM\u003c/em\u003e\u003csub\u003eFL\u003c/sub\u003e were separately ligated into pXZ2\u003csup\u003e19\u003c/sup\u003e that was cut with NdeI-EcoRI, yielding pXZ35 and pXZ36 (see Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; harboring plasmid pSET152, pXZ35 and pXZ36 were used to assess the membrane topology of StlP, which were performed as described previously\u003csup\u003e27\u003c/sup\u003e. Full-length BlaM (BlaM\u003csub\u003eFL\u003c/sub\u003e) expressed from pXZ36 served as a control for \u0026beta;-lactamase activity in the medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of CWD cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCulturing and filtration of CWD cells of \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eKitasatospora\u003c/em\u003e strains was essentially performed as described\u003csup\u003e8\u003c/sup\u003e, with the exception that \u003cem\u003eS. coelicolor\u003c/em\u003e strains were grown for 16 h, while \u003cem\u003eK. viridifaciens\u003c/em\u003e strains were grown for 40 h. Quantification of CWD cells was performed with a Bright-Line\u0026trade; Hemocytometer (Merck), as described\u003csup\u003e56\u003c/sup\u003e. Briefly, 10 \u0026micro;l of filtered culture supernatant were loaded into the counting chamber, after which cells were quantified under a Zeiss Axio microscope equipped with an Axiocam 105 camera. CWD cell numbers were counted manually, and the density was calculated. For each strain, the measurements were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo purify the stomatin-encoding domain of StlP (StlP\u003csub\u003eSD\u003c/sub\u003e), \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells harboring plasmid pXZ17 were cultured at 37\u0026deg;C to an OD\u003csub\u003e600\u003c/sub\u003e of 0.8 in LB medium containing 50 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin. Then, 0.5 mM isopropyl \u0026beta;-D-thiogalactopyranoside was added to induce protein expression, after which cells were grown at 30\u0026deg;C for 18 h. The induced cells were subsequently lysed by sonication in binding buffer (50 mM Tris\u0026ndash;HCl, 200 mM NaCl, pH 8.0), and after centrifugation, the lysate was loaded on a Ni\u003csup\u003e2+\u003c/sup\u003e-chelating column equilibrated with binding buffer. Ten column volumes of washing buffer (50 mM Tris\u0026ndash;HCl, 200 mM NaCl, 0.1 mM imidazole, pH 8.0) and 5 mL of elution buffer (50 mM Tris\u0026ndash;HCl, 200 mM NaCl, 10 mM imidazole, pH 8.0) were used to wash and elute StlP\u003csub\u003eSD\u003c/sub\u003e, respectively. Finally, the protein was purified by gel filtration using a Hiload 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with buffer (50 mM Tris\u0026ndash;HCl, 100 mM NaCl, pH 8.0). Sample fractions were analyzed on a 12.5% SDS-PAGE gel. Fractions were stored directly at -80\u0026deg;C or first concentrated to 1 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with the 3 kDa molecular weight cutoff concentrator (Millipore) and then stored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical cross-linking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cross-linking of StlP\u003csub\u003eSD\u003c/sub\u003e with glutaraldehyde (GA, 50% in H\u003csub\u003e2\u003c/sub\u003eO, Sigma) was performed as described\u003csup\u003e44\u003c/sup\u003e with the following modifications. Briefly, 1 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of StlP\u003csub\u003eSD\u003c/sub\u003e was treated with 0.02, 0.05 or 0.1% GA for 10 or 60 min at room temperature in 50 \u0026micro;l buffer (50 mM Tris\u0026ndash;HCl, 100 mM NaCl, pH 8.0). Reactions were quenched with 0.2 M glycine-NaOH (pH 9.5) for 5 min at room temperature. Detection of cross-linked protein was performed by loading the samples on a 12.5% SDS-PAGE gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize the emergence of CWD cells, spores of the wild-type strain and the ∆\u003cem\u003estlP\u003c/em\u003e mutant were pre-germinated in double strength germination medium\u003csup\u003e46\u003c/sup\u003e. Then 10 \u0026micro;l of germlings were used for live-imaging, for which an ibiTreat 35 mm low imaging dish (ibidi) and a LPMA-pad was used as before\u003csup\u003e8\u003c/sup\u003e. Live imaging was carried out using a Zeiss LSM900 Airyscan 2 microscope. If necessary, Z-Stack acquisitions were used. For visualization of membrane and nucleic acids, 0.05 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FM5-95 and 0.5 \u0026micro;M SYTO-9 (Sigma) were used, respectively.\u003c/p\u003e\n\u003cp\u003eThe detection of nascent peptidoglycan was done by using BODIPY-FL vancomycin (Sigma), essentially as described\u003csup\u003e57\u003c/sup\u003e. Briefly, after growing \u003cem\u003eStreptomyces\u003c/em\u003e strains in LPB medium for 16 h, mycelia were collected (3300 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 min) and resuspended in 200 \u0026micro;l of fresh LPB medium containing 1 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BODIPY FL vancomycin and 1 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e vancomycin. After 10 min incubation at 30\u0026deg;C, the mycelia were washed 3 times with PBS. 5 \u0026micro;l of the washed mycelia was used for microscopy analysis using a Zeiss LSM900 Airyscan 2 microscope.\u003c/p\u003e\n\u003cp\u003eFor visualizing fluid membrane microdomains, mycelia were stained with DilC\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e33,34\u003c/sup\u003e. Briefly, DilC\u003csub\u003e12\u003c/sub\u003e was dissolved in DMSO and 10 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e stock was prepared. For sample preparation, spores of each strain were inoculated in LPB medium at a final concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After 16h of growth, mycelia were collected by centrifuge (3300 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 min) and resuspended in prewarmed fresh LPB medium supplemented with 100 \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DilC12, followed by growth for 3 h at 30\u0026deg;C while shaking at 100 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Then, mycelia were collected, washed 3 times with prewarmed LPB supplemented with 1% DMSO, and resuspended in the same wash buffer. DilC\u003csub\u003e12\u003c/sub\u003e signal was detected via a Cy3 filter (535 nm excitation and 590 nm emission) using a Zeiss LSM900 Airyscan 2 microscope, wherein the cultivation chamber had been set 30\u0026deg;C to avoid temperature changes during imaging.\u003c/p\u003e\n\u003cp\u003eFor measurement of membrane fluidity, samples were prepared essentially as described\u003csup\u003e32\u003c/sup\u003e. Briefly, Laurdan (6-Dodecanoyl-N, N-dymethyl2-naphthylamine, Sigma) was dissolved in dimethylformamide (DMF) and a 10 mM stock was prepared. For preparation of mycelia for Laurdan staining, 20 ml 16 h-old mycelia were collected (3300 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 min) and resuspended in 1 ml 30\u0026deg;C pre-warmed LPB medium containing 1 mM Laurdan. For sample preparation of the \u003cem\u003estlP\u003c/em\u003e mutant, the culture was filtrated through a 100 \u0026micro;m cut-off filter (Falcon Cell Strainer 100 \u0026micro;m Nylon) to remove the majority of the CWD cells. The filtered mycelium was resuspended in 1 ml 30\u0026deg;C pre-warmed LPB medium and used for Laurdan staining. After 10 min incubation in the dark at 30\u0026deg;C, stained mycelia were collected again and washed twice in 30\u0026deg;C pre-warmed PBS buffer supplemented with 20% sucrose and 1% DMF, and finally resuspended in 200 \u0026micro;l pre-warmed PBS buffer supplemented with 20% sucrose. Fluorescent intensities were measured at 435 and 490 nm, following excitation at 350 nm using a Zeiss LSM900 Airyscan 2 microscope, wherein the cultivation chamber had been set 30\u0026deg;C to avoid temperature changes during imaging. To calculate the membrane fluidity at hyphal tips, the tip region was cropped as a square (1 \u0026micro;m x 1 \u0026micro;m) from the image and the corresponding generalized polarization (GP) value was determined as described\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor measurement of hyphal diameters, 16 h-old mycelia were collected (3300 rpm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 min) and resuspended in fresh LPB medium containing 0.05 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FM5-95. The distance between the stained membranes was used to measure the hyphal diameters by averaging the diameter at 3 distinct spots in the hypha.\u003c/p\u003e\n\u003cp\u003eThe cellulose-like glycan at hyphal tips was visualized by calcofluor white (Sigma) staining and quantified as previous described\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor analysis of hyphal branching patterns, mycelium was grown from single spore and imaged using a Zeiss Axio microscope equipped with an Axiocam 105 camera as described previously\u003csup\u003e19\u003c/sup\u003e. The distance from the tip to the proximal branch point was measured. A proximal branch was defined as having a length of 1\u0026ndash;4 \u0026micro;m as previous described\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor measurement of colony sizes, strain were grown on LPMA medium in petri dishes with a 9 cm diameter aiming for \u0026plusmn;\u0026thinsp;100 colonies per plate. After growing them for 5\u0026thinsp;~\u0026thinsp;7 days at 30\u0026deg;C, plates were scanned with Epson Perfection V37 scanner and colony size was measured subsequently.\u003c/p\u003e\n\u003cp\u003eMycelial Live/dead staining was performed using the LIVE/DEAD \u003cem\u003eBac\u003c/em\u003eLight\u0026trade; Bacterial viability kit (L7012; ThermoFisher) following the manufacturer\u0026rsquo;s instructions. Briefly, spores of the wild-type strain and the \u003cem\u003estlP\u003c/em\u003e mutant were streaked on LPMA medium. After growth for 16h, the excised ager pieces were inverted and positioned atop 10 \u0026micro;l mixture of SYTO-9 and propidium iodide (PI) nucleic acid stains from the kit with final concentration at 5 \u0026micro;M. Images were taken using a Zeiss LSM900 Airyscan 2 microscope after 10 mins incubation. The viability was calculated by dividing the integrated grey intensity of the fluorescence in the green channel by the integrated intensity of the fluorescence in the red channel. All measurement and images processing were executed with ImageJ software (version 2.0.0/1.53c/Java 1.8.0_172/64-bit).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSacculus isolation and Cryo-electron tomography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation of sacculi of \u003cem\u003eS. coelicolor\u003c/em\u003e M145 and the \u003cem\u003estlP\u003c/em\u003e mutant was essentially performed as described\u003csup\u003e60\u003c/sup\u003e, except that 16h-old liquid cultures were used and the step of removing teichoic acids was neglected.\u003c/p\u003e\n\u003cp\u003eSample preparation for cryo-electron tomography (cryo-ET) was performed as described\u003csup\u003e60\u003c/sup\u003e. Briefly, after adding the colloidal gold beads, sacculi solutions were vitrificated and applied on the EM grids. Grids were examined using a 120 kV Talos TEM (FEI/ThermoFisher) and cryo-ET data were collected using a Titan Krios instrument (ThermoFisher Scientific). The measurement of cell wall thickness was performed as described\u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein domains and protein structures were predicted by InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003c/span\u003e) and AlphaFold 2.0\u003csup\u003e61\u003c/sup\u003e. The prediction of protein membrane topology was performed by TMHMM (Version 2.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/service.php?TMHMM-2.0\u003c/span\u003e\u003c/span\u003e). Alignment of protein structures was done by PyMOL software (Version 2.5). Amino acids sequence alignment was done by ESPript 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo phylogenetically compare StlP with other SPFH proteins, MEGA 7 was used. Amino acid sequences of all SPFH proteins were downloaded from the UniPort database. For phylogenetic analysis of the distribution of StlP, the amino acid sequence of StlP was used to run Position-Specific Iterative (PSI)-BLAST to find homologs in the dataset of 15405 RefSeq representative bacteria and archaea. The homologs with a bitscore\u0026thinsp;\u0026gt;\u0026thinsp;130 were chosen and subsequently each hit was subjected to membrane topology prediction using TMHMM server (Version 2.0). Only hits with an identical membrane topology were considered valid StlP homologs. The phylogenetic tree was annotated using iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor statistical analyses, GraphPad Prism software (version 8.0.2) was used. Significance was determined using student\u0026rsquo;s t-test.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was funded by a Vici grant (VI.C.192.002) from the Dutch Research Council to DC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCabeen MT, Jacobs-Wagner C (2005) Bacterial cell shape. 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Nat Methods 19:679\u0026ndash;682. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41592-022-01488-1\u003c/span\u003e\u003cspan address=\"10.1038/s41592-022-01488-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"membrane microdomain, filamentous Actinobacteria, streptomyces, stomatin, StlP, polar growth, cell wall-deficient cells","lastPublishedDoi":"10.21203/rs.3.rs-3811693/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3811693/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cell wall represents an essential structure conserved among most bacteria, playing a crucial role in growth and development. While extensively studied model bacteria have provided insights into cell wall synthesis coordination, the mechanism governing polar growth in actinobacteria remains enigmatic. Here we identify the stomatin-like protein StlP as a pivotal factor essential for orchestrating polar growth in filamentous actinobacteria under hyperosmotic stress. StlP facilitates the establishment of a membrane microdomain with increased membrane fluidity, a process crucial for maintaining proper growth. The absence of StlP leads to branching of filaments, aberrant cell wall synthesis, thinning of the cell wall, and the extrusion of cell wall-deficient cells at hyphal tips. StlP interacts with key components of the apical glycan synthesis machinery, providing protection to filaments during apical growth. Introduction of StlP in actinobacteria lacking this protein enhances polar growth and resilience under hyperosmotic stress, accompanied by the formation of a membrane microdomain. Our findings imply that stomatin-like proteins, exemplified by StlP, confer a competitive advantage to actinobacteria encountering hyperosmotic stress. Given the widespread conservation of StlP in filamentous actinobacteria, our results propose that the mediation of polar growth through membrane microdomain formation is a conserved phenomenon in these bacteria.\u003c/p\u003e","manuscriptTitle":"The stomatin-like protein StlP organizes membrane microdomains to govern polar growth in filamentous actinobacteria under hyperosmotic stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-22 01:36:36","doi":"10.21203/rs.3.rs-3811693/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4ab2fb4-3157-4f38-8eb8-b7fd5d057fb0","owner":[],"postedDate":"January 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28115528,"name":"Biological sciences/Microbiology"},{"id":28115529,"name":"Biological sciences/Biochemistry"}],"tags":[],"updatedAt":"2025-03-19T07:09:00+00:00","versionOfRecord":{"articleIdentity":"rs-3811693","link":"https://doi.org/10.1038/s41467-025-58093-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-18 04:00:00","publishedOnDateReadable":"March 18th, 2025"},"versionCreatedAt":"2024-01-22 01:36:36","video":"","vorDoi":"10.1038/s41467-025-58093-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58093-x","workflowStages":[]},"version":"v1","identity":"rs-3811693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3811693","identity":"rs-3811693","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}