The development of stimuli-responsive liposomes paves the way for targeted and controlled drug delivery. This includes thermoresponsive liposomes, in which increased temperature can promote bilayer destabilization. This thermoresponsiveness can be achieved by incorporating lipids with defined melting transitions or LCST-type polymers that are anchored to the membrane. In this study, we developed nanometric liposomes decorated with lipopolyproline, an LCST-type polymer anchored to the membrane. These systems, termed polyprolinated liposomes, were characterized by cryoSEM, cryoTEM, DLS, and 2H and 31P solid-state NMR to highlight the temperature-dependent changes in surface properties and morphology. Quantitative measurement of sulforhodamine B release demonstrated increased membrane permeability with temperature changes, highlighting the potential of polyprolinated liposomes as thermoresponsive drug delivery systems or drug reservoirs in multicompartmental systems.

The controlled assembly of extracellular filaments is essential for bacterial multicellularity and surface colonization. While Gram-positive bacteria rely on a variety of mechanisms to construct surface-associated fibers, many noncanonical pathways remain largely unexplored. Here, we identify a regulated, sortase-independent system in Bacillus cereus that governs the polymerization of filaments within the extracellular matrix (ECM). This tripartite system comprises CapP, a chaperone-like protein, and the structural subunits TasA and CalY. CapP modulates filament formation in a concentration- and domain-dependent manner, promoting ordered heteropolymer assembly while preventing uncontrolled aggregation. Disrupting this pathway leads to distinct compensatory changes in matrix composition-including exopolysaccharide expression, extracellular DNA release, and flagellar regulation-revealing an unexpected level of matrix plasticity. Our findings uncover a unique mechanism of ECM biogenesis in Gram-positive bacteria and suggest that plasticity in matrix organization may be a widespread adaptive strategy across bacterial lineages.

The one-bond proton-carbon coupling constant (1JCH) is an insightful probe of carbohydrate configuration. Equatorial and axial protons at the C1 position typically exhibit distinct 1JCH values, enabling NMR measurements to distinguish α- and β-configurations in carbohydrates. In principle, such measurements could provide insights into carbohydrates in the cell walls of intact microbes. However, traditionally, these measurements are performed by solution NMR with carbohydrates that were extracted, solubilized and fractionated, leaving the biological relevance of the measurements uncertain. Here, we demonstrate that 1H-detected solid-state NMR with fast magic-angle spinning allows quantitative measurements of 1JCH couplings for mobile capsular polysaccharides, directly on submilligram amounts of pathogenic cells. Our approach is demonstrated on intact cells of the pathogenic yeast Cryptococcus neoformans. High-resolution proton-detected spectra enabled the determination of coupling constants for five mobile polysaccharide units of the cryptococcal capsule, revealing their native configurations and confirming previous solution NMR-based anomeric configuration assignments.

The bacterial actin homolog MreB plays a key role in rod cell shape determination. We recently showed that MreB from the Gram-positive bacterium Geobacillus stearothermophilus (MreBGs) polymerizes into straight pairs of protofilaments in the presence of both ATP and a lipid surface. Membrane interaction is thought to be mediated by electrostatic interactions with anionic lipids, with final anchoring relying on two spatially close hydrophobic motifs that protrude from the MreBGs monomers, forming a putative membrane-insertion domain. Here, we determined the binding properties of ATP and ADP to MreBGs using fluorescence anisotropy and monitored ATP-mediated binding and polymer formation on lipid bilayers using liposome-binding assays and atomic force microscopy, respectively. Finally, we used solid-state NMR to visualize the interaction between the membrane and MreBGs at the atomic level. Our findings reveal that divalent cations are required for nucleotide binding and that, unlike eukaryotic actin, MreBGs has similar affinity for both ATP and ADP. We also show that monomeric MreBGs establishes peripheral contacts with the membrane likely through electrostatic interactions, while Mg⋅ATP-induced MreBGs filaments insert into the lipid bilayer without interfering with the membrane lamellar phase and have a significant local fluidifying effect.

The development of stimuli-responsive liposomes paves the way for targeted and controlled drug delivery. This includes thermoresponsive liposomes, in which increased temperature can promote bilayer destabilization. This thermoresponsiveness can be achieved by incorporating lipids with defined melting transitions or LCST-type polymers that are anchored to the membrane. In this study, we developed nanometric liposomes decorated with lipopolyproline, an LCST-type polymer anchored to the membrane. These systems, termed polyprolinated liposomes, were characterized by cryoSEM, cryoTEM, DLS, and 2H and 31P solid-state NMR to highlight the temperature-dependent changes in surface properties and morphology. Quantitative measurement of sulforhodamine B release demonstrated increased membrane permeability with temperature changes, highlighting the potential of polyprolinated liposomes as thermoresponsive drug delivery systems or drug reservoirs in multicompartmental systems.

Since the publication of the fluid mosaic model for membranes in 1972, numerous spectroscopic techniques, including solid-state NMR (ssNMR), have been developed to assess membrane fluidity. While model membranes made from synthetic lipids have been widely used, experiments on natural membranes have remained rare. The first demonstration that natural membranes had a micro-fluidity comparable to that of model membranes was published by Ian CP Smith’s team in the 1970s. This article reviews the first and most recent ssNMR experiments aimed at studying the dynamics of natural membranes, as measured by order parameters, and their comparison with models.

Bacillus subtilis is widely studied in the microbial secondary metabolite (SM) field due to its rich variety of important natural products and genetic tractability. We report a pigment observed in B. subtilis soil isolate MB9_B4 on certain media. We characterize the conditions where this pigment is produced and identify the corresponding biosynthetic gene cluster (BGC) using a comparative genomic approach exploiting our strain collection containing other isolates with pigment production ability. The responsible BGC carried several genes, which were annotated as parts of the tryptophan biosynthesis pathway, possibly originating from duplication and divergence of originally primary metabolism. Identification of the pigment gene cluster additionally led to the discovery of additional pigment BGC carrier B. subtilis isolates, some of which were described at the earliest in 1896 under the name Bacillus aterrimus, referring to a characteristic dark pigmentation (the Latin "aterrimus" meaning very black). In addition, we employed solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopies to characterize the chemical groups of the pigment. This study describes the chemical and biological features of a new class of SM BGC, which we hope will serve to improve the current BGC discovery pipelines in Bacilli.IMPORTANCEIdentification of novel microbial secondary metabolites (SMs) and their biosynthetic gene cluster (BGC) has become increasingly difficult, especially in Bacilli, as the tools for screening and genome mining are dependent on clear function or similarity to already known BGCs. Pigments are SMs identified by their absorption of visible light, resulting in a certain color perceived by our eyes at sufficient concentrations. Thereby, pigments provide evidence of a BGC without knowing the sequence or function. Expanding the known repertoire of SM BGCs with novel BGCs will further reinforce the identification of a broader set of BGCs by mining tools such as antiSMASH.

From archaea to mammals evolutionary conserved flotillins are scaffolding proteins, recognized for their nandomain-segregating activity. Flotillins form basket-like oligomeric architectures on the membrane, based on a conserved secondary structure composition of the monomeric subunits: a membrane-targeting region, an SPFH domain and a coiled-coil “flotillin” domain. In B. subtilis, the two flotillins FloT and FloA are present, localizing mainly in distinct nanodomains and executing multiple cellular functions. We here use deuterium and phosphorus solid-state NMR to monitor the effect of the different flotillins FloT and FloA and their structural components on model membranes. We find a clear disordering effect of FloT and FloA on the membranes reaching the carbon positions in the centre of the membrane. This effect is imposed by the hydrophobic region and the adjacent SPFH domain and, surprisingly, further supported by the membrane-distant flotillin domain. Biological implications of this disordering action are discussed.

Following the publication of biological membrane models in the 1970s, Joachim Seelig was the first to experimentally demonstrate the dynamic nature of these membranes. He conducted the first ssNMR experiments to measure the order parameters of the CD (2H) bond of lipids deuterium-labelled, showing a fairly fluid membrane interior. Since then, the order parameters of the CD, CH and CC bonds have been measured. They can be used to describe the dynamics of membranes on several space and time scales: intramolecular (Å/ns-ps), molecular (nm/ns-100 ns) and collective (membrane deformations, μm/μs). The profile of CD, CH, CC order parameters across the membrane bilayer allows us to describe the lipid membrane as being very rigid at the glycerol and chain levels and very fluid at its center and surface. This is true for lipid chains carrying double bonds, rings or branched methyl groups. Bipolar lipids that span the entire membrane do not have a very fluid membrane interior. Sterols modulate membrane dynamics, increasing order parameters in the fluid phase and decreasing them in the gel phases. They can be described as regulators of membrane dynamics, as they maintain the membrane in a dynamic state that varies very little when environmental factors change (temperature, pH, etc.). The description of order parameters by statistical mechanics allows the length of the chains, the thickness of the bilayer and the membrane elastic constants to be calculated accurately. The surface area of each lipid in the membrane can also be calculated from the plateau of order parameters (positions C3-C10): AL=831-SCDplat.

Pathogenic fungal and bacterial cells are enveloped within a cell wall, a molecular barrier at their cell surface, and a critical architecture that constantly evolves during pathogenesis. Understanding the molecular composition, structural organization, and mobility of polysaccharides constituting this cell envelope is crucial to correlate cell wall organization with its role in pathogenicity and to identify potential antifungal targets. For the fungal pathogen Cryptococcus neoformans, the characterization of the cell envelope has been complexified by the presence of an additional external polysaccharide capsular shell. Here, we investigate how magic-angle spinning (MAS) solid-state NMR techniques increase the analytical capabilities to characterize the structure and dynamics of this encapsulated pathogen. The versatility of proton detection experiments, dynamic-based filters, and relaxation measurements facilitate the discrimination of the highly mobile external capsular structure from the internal rigid cell wall of C. neoformans. In addition, we report the in situ detection of triglyceride molecules from lipid droplets based on NMR dynamic filters. Together, we demonstrate a nondestructive technique to study the cell wall architecture of encapsulated microbes using C. neoformans as a model, an airborne opportunistic fungal pathogen that infects mainly immunocompromised but also competent hosts.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5 '-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G(-2)A(-1)G(+1)U(+2) motif of the 5 '-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5 '-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A(-1) bulged 5 '-splice sites, but also identify common pharmacophores required for modulating 5 '-splice site selection with small molecules.

A perforated silica layer with structural correlation is engineered using sol–gel chemistry, applied to large-scale flat and curved surfaces.

Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.

Remorins are multifunctional proteins, regulating immunity, development and symbiosis in plants. When associating to the membrane, remorins sequester specific lipids into functional membrane nanodomains. The multigenic protein family contains six groups, classified upon their protein-domain composition. Membrane targeting of remorins occurs independently from the secretory pathway. Instead, they are directed into different nanodomains depending on their phylogenetic group. All family members contain a C-terminal membrane anchor and a homo-oligomerization domain, flanked by an intrinsically disordered region of variable length at the N-terminal end. We here combined molecular imaging, NMR spectroscopy, protein structure calculations and advanced molecular dynamics simulation to unveil a stable pre-structuration of coiled-coil dimers as nanodomain-targeting units, containing a tunable fuzzy coat and a bar code-like positive surface charge before membrane association. Our data suggest that remorins fold in the cytosol with the N-terminal disordered region as a structural ensemble around a dimeric anti-parallel coiled-coil core containing a symmetric interface motif reminiscent of a hydrophobic Leucine zipper. The domain geometry, the charge distribution in the coiled-coil remorins and the differences in structures and dynamics between C-terminal lipid anchors of the remorin groups provide a selective platform for phospholipid binding when encountering the membrane surface.

Bacillus subtilis is widely studied in the microbial secondary metabolite (SM) field due to its rich variety of important natural products and genetic tractability. However, identification of novel SMs and their biosynthetic gene clusters (BGCs), has become increasingly difficult, especially in Bacilli, as the tools for screening and genome mining are dependent on clear function or similarity to already known BGCs. Pigments are SMs identified by their absorption of visible light, resulting in a certain color perceived by our eyes at sufficient concentrations. Thereby, pigments provide the evidence of a BGC without knowing the sequence or function. Expanding the known repertoire of SM BGCs with novel BGCs will further reinforce identification of a broader set of BGCs by mining tools such as antiSMASH. Here, we study a pigment observed in B. subtilis soil isolate MB9_B4 on certain media. We characterize the conditions where this pigment is produced and identify the corresponding BGC using a comparative genomic approach exploiting our strain collection containing other isolates with pigment production ability. The responsible BGC carried several genes, which were annotated as parts of the tryptophan biosynthesis pathway, possibly originating from a duplication and divergence of an originally primary metabolism. Identification of the pigment gene cluster additionally lead to the discovery of additional pigment BGC carrier B. subtilis isolates, some of which were described at the earliest in 1896 under the name Bacillus aterrimus, with a name referring to a dark pigmentation (the Latin aterrimus meaning very black). In addition, we employed solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopies to characterize the chemical groups of the pigment. This study describes the chemical and biological features of a new class of SM BGC, which we hope will serve to improve the current BGC discovery pipelines in Bacilli. ### Competing Interest Statement The authors have declared no competing interest.

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C-13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C-13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

Diazirine is one of the smallest photo-sensitive moieties discovered to date. When incorporated in the structure of phospholipids, its minimal size has a low impact on the morphology of the resultant liposomes. A DMPC-diazirine analogue was designed and subsequently used to generate liposomes with a lower permeability and a lower phase-transition temperature compared to control DMPC liposomes. Contrary to control liposomes, in the absence of light, the photosensitive nanoparticles retained the cargo (calcein) for at least 10 days. However, upon irradiation, diazirine's conversion triggered the fluorophore release within minutes. The kinetics of the release could be tuned by the power and duration of the irradiation process. The same approach can be used on other nanomaterials, with the final goal of discovering a release profile appropriate not only for therapeutic applications, but also for agrochemicals delivery or cosmoceutics.

Fibrillary proteins are structural scaffolds that diversify the functionality of the architectural bacterial extracellular matrix. Here, we report a previously uncharacterized bacterial factor called bc1280 that is exclusive to B. cereus group and indispensable for the establishment of a biofilm lifestyle. We propose that BC1280 is an architectural conductor for the assembly of the amyloid platform, leading to the polymerization of heteropili with two functional amyloids, CalY and TasA. From its cellular localization in the cell membrane, aggregates of BC1280 nucleate the polymerization of CalY, which further incorporates TasA into nascent pili. Additionally, BC1280 modulates the expression of EPS via an uncharacterized pathway that is activated by a protease and an ECF-type sigma factor. The pilus biogenesis system described in this work partially mirrors curli system in Escherichia coli , unveiling a new paradigm in the structural biology of gram-positive bacteria and highlighting the complexity of extracellular matrix assembly in B. cereus .### Competing Interest StatementThe authors have declared no competing interest.

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short β-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.