Hydrophobic Sheath Research Articles

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

The second messenger bis-3,5-cyclic di-guanosine monophosphate (c-di-GMP) determines when Streptomyces initiate sporulation. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor σWhiG . However, functions of the development-specific diguanylate cyclases (DGCs) CdgB and CdgC, and the c-di-GMP phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5'pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA, and ∆rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved in cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores.

Expression of the chaplin and rodlin hydrophobic sheath proteins in Streptomyces venezuelae is controlled by σBldN and a cognate anti‐sigma factor, RsbN

The chaplin and rodlin proteins together constitute the major components of the hydrophobic sheath that coats the aerial hyphae and spores in Streptomyces, and mutants lacking the chaplins are unable to erect aerial hyphae and differentiate on minimal media. We have gained insight into the developmental regulation of the chaplin (chp) and rodlin (rdl) genes by exploiting a new model species, Streptomyces venezuelae, which sporulates in liquid culture. Using microarrays, the chaplin and rodlin genes were found to be highly induced during submerged sporulation in a bldN-dependent manner. Using σ(BldN) ChIP-chip, we show that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σ(BldN) . sven3186 (here named rsbN for regulator of sigma BldN), the gene lying immediately downstream of bldN, was also identified as a target of σ(BldN) . Disruption of rsbN causes precocious sporulation and biochemical experiments demonstrate that RsbN functions as a σ(BldN) -specific anti-sigma factor.

Streptomyces morphogenetics: dissecting differentiation in a filamentous bacterium

During the life cycle of the filamentous bacteria Streptomyces, morphological differentiation is closely integrated with fundamental growth and cell-cycle processes, as well as with truly complex multicellular behaviour that involves hormone-like extracellular signalling and coordination with an extraordinarily diverse secondary metabolism. Not only are the bacterial cytoskeleton and the machineries for cell-wall assembly, cell division and chromosome segregation reorganized during sporulation, but the developmental programme of these fascinating organisms also has many unusual elements, including the formation of a sporulating aerial mycelium and the production of a surfactant peptide and a hydrophobic sheath that allow cells to escape from the surface tension of the growth medium.

Ecophysiology of the filamentous Alphaproteobacterium Meganema perideroedes in activated sludge

A comprehensive study of the ecophysiology of the filamentous Meganema perideroedes affiliated to the Alphaproteobacteria, possessing a “ Nostocoida limicola Type II” filamentous morphology was conducted. This morphotype often causes serious bulking problems in activated sludge wastewater treatment plants, and hardly anything is known about its physiology. The study was carried out by applying a suite of in situ methods in an industrial activated sludge treatment plant with excessive growth of this species. The experiments revealed a very versatile organism able to take up a large variety of organic substrates under aerobic conditions. It had a remarkably high storage capacity forming polyhydroxyalkanoates from most substrates tested. When nitrate was present as e-acceptor, the number of substrates to be consumed by M. perideroedes was more restricted compared to aerobic conditions. With nitrite as e-acceptor, only acetate and glucose among the substrates tested could be assimilated and used for storage and possibly growth. This indicated that M. perideroedes might be able to denitrify under certain conditions, which is unusual for filamentous bacteria in activated sludge. No substrate uptake or storage was seen under anaerobic conditions. M. perideroedes was relatively hydrophobic, compared to other filamentous bacteria and microcolonies present in the sludge, indicating the presence of a hydrophobic sheath. Several excreted surface-associated exoenzymes were detected in the sludge, but M. perideroedes never showed any activity, except once after a breakdown in the production facility. This confirmed that M. perideroedes mainly grows on soluble substrates. Based on the studies of the ecophysiology of M. perideroedes, potential control strategies are suggested.

Exploration of synergism between a polymer matrix and gold nanoparticles for selective determination of dopamine

In this work, synergism between the conducting polymer matrix and gold nanoparticles is explored for dopamine sensing in the presence of excess ascorbic acid using poly (3,4-ethylenedioxythiophene) (PEDOT) in neutral phosphate buffer solutions (PBS 7.4). The electrodeposited thin films of PEDOT were characterized by atomic force microscopy (AFM) (and current sensing-AFM), and the incorporated Au nanoparticles by UV–Vis spectroscopy and electrochemical methods. Distinct “oxidized” and “reduced” regions of the polymer film were revealed in the CS-AFM. Electrochemical studies have shown that the catalytic oxidation of DA and AA on PEDOT modified electrodes can afford a peak potential separation of ∼230 mV. Further enhancement in the oxidation current for DA/AA oxidation was achieved by incorporation of Au nanoparticles in the PEDOT modified electrodes. The nanometer sized gold particles favour the nanomolar sensing of dopamine in the presence of excess of ascorbic acid (1.0 mM). The DA oxidation current increases linearly with DA concentration and the detection limit of DA is found to be ∼2 nM. Amperometric detection under stirred conditions is free from electrode fouling at lower concentrations of DA and is only slightly affected at concentrations beyond 20 μM. The combined effect of Au nanoparticles and the PEDOT matrix is based on the Au nano surrounded by a “hydrophobic sheath” tending to reside within these hydrophobic regions of PEDOT, increasing the DA oxidation current.

The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.

Lamellar Langmuir–Blodgett films of hydrophobized colloidal gold nanoparticles by organization at the air–water interface

The organization of hydrophobically modified colloidal gold nanoparticles at the air–water interface and the formation thereafter of lamellar, multilayer films of the nanoparticles by the Langmuir–Blodgett (LB) technique is described in this paper. The hydrophobization of the gold colloidal particles was accomplished by the electrostatic extraction of carboxylic acid derivatized gold particles (synthesized in an aqueous medium, 35 Å in size) from solution into thermally evaporated fatty amine films by a simple immersion procedure. The acid–base complex formed by the association of the carboxylic acid groups bound to the colloidal particle surface and the amine groups in the lipid matrix leads to the formation of a strongly-bound hydrophobic sheath of fatty amine molecules around the particles. The colloidal gold particles can thereafter be dissolved in different organic solvents, dried and redispersed repeatedly without significant aggregation of the gold particles. The hydrophobic gold particles were dissolved in a spreading solvent and organized on the surface of water. The organization of the particles and the formation of multilayer films by the Langmuir–Blodgett technique was followed by surface pressure–area isotherm measurements of the colloidal particle Langmuir monolayer, quartz crystal microgravimetry, ultraviolet-vis spectroscopy and Fourier transform infrared spectroscopy. It was observed that a close-packed monolayer of the colloidal particles was formed on the surface of water and that excellent multilayer films of the colloidal nanoparticles can be grown on different supports by sequential transfer by the LB technique.

Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora.

The surface of many fungal spores is covered by a hydrophobic sheath termed the rodlet layer. We have determined that the rodlet protein of Neurospora crassa is encoded by a cloned gene designated bli-7, and that bli-7 is identical to the known gene eas (easily wettable). Using eas DNA as a probe we show that eas mRNA is abundant in illuminated mycelia and conidiophores but is not detectable or is barely detectable in dark-grown mycelia, mature macroconidia, microconidia, and ascospores. Mutations in the genes acon-2, acon-3, and fl block early conidiophore development; of these, only fl prevents normal eas transcription. The EAS protein is homologous to the rodlet protein (RodA) of Aspergillus nidulans, and the hydrophobins of Schizophyllum commune. eas is the first cloned conidiation (con) gene of N. crassa that is associated with a phenotypic alteration.

The sequence of the mu transmembrane segment determines the tissue specificity of the transport of immunoglobulin M to the cell surface.

Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.

Crystalline Inclusion Compounds of 2,2′-Dihydroxy-1,1′-binaphthyl with Alkali Metal Hydroxides and Ammonia

Abstract 2,2′-Dihydroxy-1,1′-binaphthyl, C20H14O2 from crystalline inclusion compounds of stoichiometries 1\frac12C20H14O2·3MOH·8H2O (M = Li,Na,K), C20H14O2·CsOH·6H2O, and 2C20H14O2·2NH3·CH3OH. In the crystal structure of the NH3 complex, the three molecular components are interlinked by hydrogen bonds to form a column with a hydrophilic stem and a hydrophobic sheath.