PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK.

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.

Light dependent synthesis of a nucleotide second messenger controls the motility of a spirochete bacterium.

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and in functions such as virulence and biofilm formation, mainly via gene regulation. Here, we show that the swimming motility of the soil bacterium Leptospira kobayashii is rapidly modulated by light stimulation. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis identified the novel sensory gene, and its expression in Escherichia coli through codon optimization elucidated the light-dependent synthesis of cyclic adenosine monophosphate (cAMP). GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. These findings suggest a new role for cAMP in rapidly controlling the flagella-dependent motility of Leptospira and highlight the global distribution of the newly discovered photoactivated cyclase among diverse microbial species.

The role of the "Thiodendron" consortium in postulating the karyomastigont chimaera of the endosymbiosis theory by Lynn Margulis.

The endosymbiosis theory of the origin of eukaryotic cell was first proposed more than a hundred years ago. In the second half of the 20th century, Lynn Margulis suggested a new interpretation of the origin of the nucleus in modern eukaryotes. The background was the study of the consortium "Thiodendron", a symbiotic bacterial community, which includes anaerobic aerotolerant motile spirochaetes and sulfidogenic bacteria (sulfidogens) of vibrioid form with a fermentation type of metabolism. Spirochaetes supply sulfidogens with metabolites (pyruvate and, probably, organic nitrogenous products of cell lysis) and get hydrogen sulfide from sulfidogens that helps to maintain a low redox potential. At low oxygen concentrations, spirochaetes are able to assimilate glucose more efficiently. Margulis hypothesized about the symbiotic origin of the nucleus by adding the bacterium Spirochaeta to the Thermoplasma-like archaea. She considered the "Thiodendron"-like consortium to be an intermediate stage in evolution. According to Margulis, the conversion of carbohydrates and the oxidation of Н2S to S0 by the bacterium provided the archaea with electron acceptors for anaerobic respiration, as shown for modern thermoplasmas and products saturated with carbon. The use of carbon sources increased by attaching the floating bacterium to the archaea. More efficient microaerobic oxidation of glucose pre-adapted the spirochaetes for association with Thermoplasma. However, modern "Thiodendron"-like consortia are not in stable symbiosis and a sulfidogenic component of the consortium is capable for fermentation, rather than anaerobic respiration, which makes the theory by Margulis disputable.

Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel.

SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.

Implications of back-and-forth motion and powerful propulsion for spirochetal invasion.

The spirochete Leptospira spp. can move in liquid and on a solid surface using two periplasmic flagella (PFs), and its motility is an essential virulence factor for the pathogenic species. Mammals are infected with the spirochete through the wounded dermis, which implies the importance of behaviors on the boundary with such viscoelastic milieu; however, the leptospiral pathogenicity involving motility remains unclear. We used a glass chamber containing a gel area adjoining the leptospiral suspension to resemble host dermis exposed to contaminated water and analyzed the motility of individual cells at the liquid-gel border. Insertion of one end of the cell body to the gel increased switching of the swimming direction. Moreover, the swimming force of Leptospira was also measured by trapping single cells using an optical tweezer. It was found that they can generate [Formula: see text] 17 pN of force, which is [Formula: see text] 30 times of the swimming force of Escherichia coli. The force-speed relationship suggested the load-dependent force enhancement and showed that the power (the work per unit time) for the propulsion is [Formula: see text] 3.1 × 10-16 W, which is two-order of magnitudes larger than the propulsive power of E. coli. The powerful and efficient propulsion of Leptospira using back-and-forth movements could facilitate their invasion.

Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy.

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.

Treatment of anthraquinone dye textile wastewater using anaerobic dynamic membrane bioreactor: Performance and microbial dynamics.

The performance and microbial community structure of anaerobic dynamic membrane bioreactor (AnDMBR) treating textile wastewater was investigated. The reactor showed excellent soluble COD and color removal of 98.5% and >97.5%, respectively. Dynamic membrane layer grown over the 3D printed dynamic membrane support showed decent rejection for high molecular weight compounds (>20 kDa); and the total suspended solid rejection by the dynamic layer was >98.8%. Gel permeation chromatography analysis of extracellular polymeric substance (EPS) and effluent samples revealed EPS accounted for more than 76.7% of low molecular weight fractions (<20 kDa) that end up in the effluent. Higher applied flux facilitated the rapid formation dynamic layer which enabled a satisfactory effluent quality. Microbial community analysis revealed that during the operation the archaeal community was relatively stable while obvious changes took place in the bacterial community. Introduction of dye Remazol Brilliant Blue R (RBBR) to the AnDMBR increased the abundances of phyla of Proteobacteria and Spirochaetae whereas fractions of Firmicutes and Euryarchaeota decreased obviously. Furthermore, relative stable abundances of phyla Aminicenantes, Bacteroidetes, Thermotogae and Chloroflexi among the top six phyla detected in the system ensured a healthy anaerobic degradation environment for RBBR wastewater treatment.

An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel.

Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated into mechanical work to modulate the open/closed probability of the pore, i.e., gating. Despite the recent advances in structural determination of CNG channels, the conformational changes associated with gating remain unknown. Here we examine the conformational dynamics of a prokaryotic homolog of CNG channels, SthK, using high-speed atomic force microscopy (HS-AFM). HS-AFM of SthK in lipid bilayers shows that the CNBDs undergo dramatic conformational changes during the interconversion between the resting (apo and cGMP) and the activated (cAMP) states: the CNBDs approach the membrane and splay away from the 4-fold channel axis accompanied by a clockwise rotation with respect to the pore domain. We propose that these movements may be converted by the C-linker to pull the pore helices open in an iris diaphragm-like mechanism.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility.

Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)(1). These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space(2-11). The universal joint or hook that links the rotary motor to the filament is composed of ∼120-130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex(9,12-17). In other bacteria, the hook can be readily dissociated by treatments such as heat(18). In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking(12,13,17). Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes.

Structural basis for cellulose binding by the type A carbohydrate-binding module 64 of Spirochaeta thermophila.

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate-binding module 64 (CBM64) appended at the C-terminus. CBM64 adsorbs to various ß1-4-linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly-roll-like fold corresponding to a surface-binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of ß-linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855-858. © 2016 Wiley Periodicals, Inc.

Microbial reduction and precipitation of vanadium (V) in groundwater by immobilized mixed anaerobic culture.

Vanadium is an important contaminant impacted by natural and industrial activities. Vanadium (V) reduction efficiency as high as 87.0% was achieved by employing immobilized mixed anaerobic sludge as inoculated seed within 12h operation, while V(IV) was the main reduction product which precipitated instantly. Increasing initial V(V) concentration resulted in the decrease of V(V) removal efficiency, while this index increased first and then decreased with the increase of initial COD concentration, pH and conductivity. High-throughput 16S rRNA gene pyrosequencing analysis indicated the decreased microbial diversity. V(V) reduction was realized through dissimilatory reduction process by significantly enhanced Lactococcus and Enterobacter with oxidation of lactic and acetic acids from fermentative microorganisms such as the enriched Paludibacter and the newly appeared Acetobacterium, Oscillibacter. This study is helpful to detect new functional species for V(V) reduction and constitutes a step ahead in developing in situ bioremediations of vanadium contamination.

Evidence of syntrophic acetate oxidation by Spirochaetes during anaerobic methane production.

To search for evidence of syntrophic acetate oxidation by cluster II Spirochaetes with hydrogenotrophic methanogens, batch reactors seeded with five different anaerobic sludge samples supplemented with acetate as the sole carbon source were operated anaerobically. The changes in abundance of the cluster II Spirochaetes, two groups of acetoclastic methanogens (Methanosaetaceae and Methanosarcinaceae), and two groups of hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales) in the reactors were assessed using qPCR targeting the 16S rRNA genes of each group. Increase in the cluster II Spirochaetes (9.0±0.4-fold) was positively correlated with increase in hydrogenotrophic methanogens, especially Methanomicrobiales (5.6±1.0-fold), but not with acetoclastic methanogens. In addition, the activity of the cluster II Spirochaetes decreased (4.6±0.1-fold) in response to high hydrogen partial pressure, but their activity was restored after consumption of hydrogen by the hydrogenotrophic methanogens. These results strongly suggest that the cluster II Spirochaetes are involved in syntrophic acetate oxidation in anaerobic digesters.

Novel family of carbohydrate-binding modules revealed by the genome sequence of Spirochaeta thermophila DSM 6192.

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative ß-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, ß-glucanase, ß-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

Spirochaeta perfilievii sp. nov., an oxygen-tolerant, sulfide-oxidizing, sulfur- and thiosulfate-reducing spirochaete isolated from a saline spring.

A novel strain of fermenting, aerotolerant, chemo-organoheterotrophic spirochaete designated P(T) was isolated from a sulfur 'Thiodendron' mat in a saline spring at the Staraya Russa resort (Novgorod Region, Russia). Cells of strain P(T) exhibited a helical shape. The spirochaete required sulfide in the growth medium and was able to oxidize it non-enzymically to elemental sulfur via the interaction of H(2)O(2) with sulfide and deposit it in the periplasmic space. Growth occurred at 4-32 °C (optimum at 28-30 °C), pH 6.0-8.5 (optimum pH 7.0-7.5), and in 0.1-1 M NaCl (optimum 0.35 M). The isolate used several sugars and polysaccharides as carbon or energy sources but did not use peptides, amino acids, organic acids or alcohols. The products of glucose fermentation were formate, acetate, ethanol, pyruvate, CO(2) and H(2). The genomic DNA G+C content was 41.7 mol%. 16S rRNA gene sequence analysis showed that strain P(T) fell within a group of species in the genus Spirochaeta, including Spirochaeta litoralis, S. isovalerica and S. cellobiosiphila, with which it shared less then 89 % sequence similarity. On the basis of its morphology, physiology and other phenotypic properties, as well as its phylogenetic position, the new isolate is considered to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta perfilievii sp. nov. is proposed. The type strain is P(T) (=DSM 19205(T) =VKM B-2514(T)).

Characterization of the cell surface glycolipid from Spirochaeta aurantia.

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Hydrogen production by anaerobic microbial communities exposed to repeated heat treatments.

Biological hydrogen production by anaerobic mixed communities was studied in laboratory-scale bioreactors using sucrose as the substrate. A bioreactor in which a fraction of the return sludge was exposed to repeated heat treatments performed better than a control bioreactor without repeated heat treatment of return sludge and produced a yield of 2.15 moles of hydrogen per mole of sucrose, with 50% hydrogen in the biogas. Terminal restriction fragment length polymorphism analysis showed that two different Clostridium groups (comprised of one or more species) were dominant during hydrogen production. The relative abundance of two other non-Clostridium groups increased during periods of decreased hydrogen production. The first group consisted of Bifidobacterium thermophilum, and the second group included one or more of Bacillus, Melissococcus, Spirochaeta, and Spiroplasma spp.

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation.

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.

[The role of oxygen in the regulation of the metabolism of aerotolerant spirochetes, a major component of "Thiodendron" bacterial sulfur mats]. / Rol' kisloroda v reguliatsii metabolizma aérotolerantnykh spirokhet--osnovnogo komponenta bakterial'nykh sernykh matov "Thiodendron".

Two spirochete strains isolated earlier from "Thiodendron" bacterial sulfur mats grew better under microaerobic (0.3-0.5 mg O2/l) than under anaerobic conditions. The microaerobic growth of these strains was accompanied by a twofold increase in the cell yield and the efficiency of glucose utilization, despite an amount of ATP (and hence glucose) was spent in this case for the synthesis of exopolysaccharides. Glucose metabolism under microaerobic conditions gave rise to more oxidized products (acetate and carbon dioxide) than under anaerobic conditions (formate, ethanol, pyruvate, and hydrogen). The paper considers two putative mechanisms implemented by aerotolerant spirochetes: adaptive (the use of a more efficient pathway of glucose catabolism) and protective (an enhanced synthesis of exopolysaccharides and the reduction of hydrogen peroxide by the reduced sulfur compounds thiosulfate and sulfide, yielding elemental sulfur). The formation of "Thiodendron" bacterial sulfur mats in saltwater environments is also discussed.

Analysis of the ability of spirochete species associated with relapsing fever, avian borreliosis, and epizootic bovine abortion to bind factor H and cleave c3b.

Some Borrelia species associated with Lyme disease bind the complement-regulatory protein factor H (fH), a process that may aid in immune evasion. In this report we demonstrate that some Borrelia species associated with relapsing fever bind fH, but not those associated with avian borreliosis and epizootic bovine abortion. Cell-bound fH was also found to mediate cleavage of exogenously supplied human C3b, demonstrating the biological relevance of fH binding and its possible importance in the pathogenesis of the relapsing-fever spirochetes.