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1.
Appl Microbiol Biotechnol ; 105(3): 1133-1145, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33427929

RESUMO

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates-soluble oligosaccharides. This issue was addressed in the current study using enzyme engineering and an enzyme evolution approach. To this end, a hypothetical endoglucanase from a thermostable bacterium Spirochaeta thermophila was for the first time cloned and characterized. The wild-type enzyme was used as a starting point for mutagenesis and molecular evolution toward a preference for the higher molecular weight substrates. The best of the evolved enzymes was more active than the wild-type enzyme toward high molecular weight substrate at temperatures below 45 °C (3-fold more active at 30 °C) and showed little or no activity with low molecular weight substrates. These findings can be instrumental in bioeconomy sectors, such as second-generation biofuels and biomaterials from lignocellulosic biomass. KEY POINTS: • A new thermostable endoglucanase was characterized. • The substrate specificity of this endoglucanase was changed by means of genetic engineering. • A mutant with a preference for long molecular weight substrate was obtained and proposed to be beneficial for cellulose fiber modification.


Assuntos
Celulase , Celulase/genética , Celulase/metabolismo , Celulose , Estabilidade Enzimática , Hidrólise , Spirochaeta , Especificidade por Substrato
2.
Biosystems ; 200: 104322, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33309968

RESUMO

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.


Assuntos
Archaea/metabolismo , Evolução Biológica , Eucariotos/metabolismo , Spirochaeta/metabolismo , Simbiose , Anaerobiose , Metabolismo dos Carboidratos , Núcleo Celular/metabolismo , Células Eucarióticas/metabolismo , Glucose/metabolismo , Sulfeto de Hidrogênio/metabolismo , Modelos Biológicos , Oxirredução , Oxigênio/metabolismo , Enxofre/metabolismo
3.
Nat Commun ; 11(1): 6401, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328472

RESUMO

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.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Spirochaeta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Ciclosporina/farmacologia , Ativação do Canal Iônico/fisiologia , Isomerismo , Cinética , Modelos Moleculares , Peptidilprolil Isomerase/metabolismo , Prolina/metabolismo
4.
Acta Crystallogr D Struct Biol ; 76(Pt 11): 1104-1113, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135681

RESUMO

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Carboidratos Epimerases/química , Spirochaeta/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Carboidratos Epimerases/genética , Domínio Catalítico , Isomerismo , Mutagênese Sítio-Dirigida , Especificidade por Substrato
5.
Sci Rep ; 10(1): 17053, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051478

RESUMO

Spirochetal bacteria were successfully isolated from mosquitoes (Culex pipiens, Aedes cinereus) in the Czech Republic between 1999 and 2002. Preliminary 16S rRNA phylogenetic sequence analysis showed that these strains differed significantly from other spirochetal genera within the family Spirochaetaceae and suggested a novel bacterial genus in this family. To obtain more comprehensive genomic information of these isolates, we used Illumina MiSeq and Oxford Nanopore technologies to sequence four genomes of these spirochetes (BR151, BR149, BR193, BR208). The overall size of the genomes varied between 1.68 and 1.78 Mb; the GC content ranged from 38.5 to 45.8%. Draft genomes were compared to 36 publicly available genomes encompassing eight genera from the class Spirochaetes. A phylogeny generated from orthologous genes across all taxa and the percentage of conserved proteins (POCP) confirmed the genus status of these novel spirochetes. The genus Entomospira gen. nov. is proposed with BR151 selected as type species of the genus. For this isolate and the closest related isolate, BR149, we propose the species name Entomospira culicis sp. nov. The two other isolates BR208 and BR193 are named Entomospira nematocera sp. nov. (BR208) and Entomospira entomophilus sp. nov. (BR193). Finally, we discuss their interesting phylogenetic positioning.


Assuntos
Spirochaetales/classificação , Spirochaetales/genética , Spirochaetales/isolamento & purificação , Animais , Artrópodes/genética , Técnicas de Tipagem Bacteriana/métodos , Composição de Bases/genética , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Spirochaeta/genética
6.
Int J Syst Evol Microbiol ; 70(12): 6373-6380, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33125316

RESUMO

A novel obligately anaerobic spirochete strain K2T was isolated from bottom marine sediments at Crater Bay of Yankicha Island (Kuril Islands, Russia). Strain K2T had helical shape and Gram-negatively stained. The optimal growth conditions were as follows: the optimum temperature was 28-30 °C with range 5-34 °C; optimal pH at 7.0-7.5 with range of 6.8-8.5; NaCl optimum at 3-3.5 % (w/v) and range of 1-7 % (w/v). Strain K2T was catalase- and oxidase-negative. Glucose fermentation products were acetate, lactate, ethanol, CO2, H2. The major fatty acids were C14 : 0, iso-C13 : 0, iso-C15:0, C14 : 0 DMA, iso-C15 : 0 DMA. The G+C content of genomic DNA was 43.2 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain K2T belonged to the genus Oceanispirochaeta of the family Spirochaetaceae. The 16S rRNA gene sequence similarity of strain K2T and O. litoralis DSM 2029T and O. sediminicola DSM 104770T was 96 and 94 %, respectively. Based on the results of our study, we propose the name Oceanispirochaeta crateris sp. nov.; type strain K2T (=DSM 16308T=VKM B-3266T). Also, the taxonomic status of Spirochaeta perfilevii was revised: 16S rRNA genes sequence showed less than 89 % similarity to nearest phylogenetic neighbours. Therefore, we proposed to separate this species into a novel genus Thiospirochaeta - T. perfilievii gen. nov., comb. nov.


Assuntos
Sedimentos Geológicos/microbiologia , Filogenia , Água do Mar/microbiologia , Spirochaetaceae/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , RNA Ribossômico 16S/genética , Federação Russa , Análise de Sequência de DNA , Spirochaeta/classificação , Spirochaetaceae/isolamento & purificação
7.
BMC Microbiol ; 20(1): 293, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32993514

RESUMO

BACKGROUND: Dental implants have become well-established in oral rehabilitation for fully or partially edentulous patients. However, peri-implantitis often leads to the failure of dental implants. The aim of this study was to understand the core microbiome associated with peri-implantitis and evaluate potential peri-implantitis pathogens based on canine peri-implantitis model. RESULTS: In this study, three beagle dogs were used to build peri-implantitis models with ligature-induced strategy. The peri-implant sulcular fluids were collected at four different phases based on disease severity during the peri-implantitis development. Microbial compositions during peri-implantitis development were monitored and evaluated. The microbes were presented with operational taxonomic unit (OTU) classified at 97% identity of the high-throughput 16S rRNA gene fragments. Microbial diversity and richness varied during peri-implantitis. At the phylum-level, Firmicutes decreased and Bacteroides increased during peri-implantitis development. At the genus-level, Peptostreptococcus decreased and Porphyromonas increased, suggesting peri-implantitis pathogens might be assigned to these two genera. Further species-level and co-occurrence network analyses identified several potential keystone species during peri-implantitis development, and some OTUs were potential peri-implantitis pathogens. CONCLUSION: In summary, canine peri-implantitis models help to identify several potential keystone peri-implantitis associated species. The canine model can give insight into human peri-implantitis associated microbiota.


Assuntos
Interface Osso-Implante/microbiologia , Implantes Dentários/microbiologia , Microbiota/genética , Peri-Implantite/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Bacteroides/classificação , Bacteroides/genética , Bacteroides/isolamento & purificação , Interface Osso-Implante/patologia , Modelos Animais de Doenças , Cães , Firmicutes/classificação , Firmicutes/genética , Firmicutes/isolamento & purificação , Variação Genética , Humanos , Ligadura/efeitos adversos , Masculino , Peptostreptococcus/classificação , Peptostreptococcus/genética , Peptostreptococcus/isolamento & purificação , Peri-Implantite/etiologia , Peri-Implantite/patologia , Filogenia , Porphyromonas/classificação , Porphyromonas/genética , Porphyromonas/isolamento & purificação , RNA Ribossômico 16S/genética , Spirochaeta/classificação , Spirochaeta/genética , Spirochaeta/isolamento & purificação
8.
Sci Rep ; 10(1): 13937, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811890

RESUMO

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.


Assuntos
Leptospira/metabolismo , Movimento/fisiologia , Infecções por Spirochaetales/metabolismo , Fenômenos Biofísicos/fisiologia , Flagelos/fisiologia , Leptospira/patogenicidade , Movimento (Física) , Pinças Ópticas , Spirochaeta/metabolismo , Spirochaeta/patogenicidade , Spirochaetales/metabolismo , Spirochaetales/patogenicidade , Fatores de Virulência
9.
Proc Natl Acad Sci U S A ; 117(20): 10839-10847, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358188

RESUMO

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.


Assuntos
Sítio Alostérico/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Spirochaeta/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Nucleotídeos Cíclicos , Conformação Proteica
10.
Bioresour Technol ; 304: 123024, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32086035

RESUMO

In present study, evaluate the effect of fine coal gasification slag (FCGS) as additive on abundance of bacterial diversity during pig manure composting. The six different dosages of FCGS 0% (T1), 2% (T2), 4% (T3), 6% (T4), 8% (T5) and 10% (T6) (dry weight basis) were mixed with original raw materials for 42 days an aerobic composting. The results indicated that FCGS adopted could affect the succession of bacterial diversity in different ways. Among all treatments, Firmicutes, Proteobacteria, Tenericutes, unidentified_Bacteria, and Actinobacteria were the highest abundance in weighted unifrac distance but Firmicutes; Proteobacteria, Actinobacteria, Bacteroidetes, and Spirochaetes were main bacteria in unweighted unifrac distance. The ß-diversity and principal component analysis indicated a significant difference in bacterial diversity in all treatments which T4 obtained difference obviously. Therefore, the results showed that T4 was a potential candidate to enhance significantly abundance of bacterial community in PM compost.


Assuntos
Compostagem , Spirochaeta , Animais , Bactérias , Carvão Mineral , Esterco , Solo , Suínos
11.
Chemosphere ; 238: 124539, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31470310

RESUMO

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.


Assuntos
Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Reatores Biológicos/microbiologia , Membranas Artificiais , Têxteis , Águas Residuárias/química , Anaerobiose , Corantes/isolamento & purificação , Corantes/metabolismo , Matriz Extracelular de Substâncias Poliméricas/microbiologia , Proteobactérias/metabolismo , Spirochaeta/metabolismo , Poluentes Químicos da Água/isolamento & purificação , Poluentes Químicos da Água/metabolismo
12.
Nat Commun ; 9(1): 3978, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266906

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Ativação do Canal Iônico , Conformação Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , AMP Cíclico/química , AMP Cíclico/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Microscopia de Força Atômica/métodos , Modelos Moleculares , Ligação Proteica , Rotação , Spirochaeta/metabolismo
13.
Biochemistry ; 57(35): 5218-5229, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30106565

RESUMO

In an effort to evaluate whether a recently reported putative metallo-ß-lactamase (MßL) contains a novel MßL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not ß-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MßLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MßLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of ß-lactam antibiotics. This study reveals a novel metal binding site in MßLs and suggests that the targeting of metal binding sites in MßLs with inhibitors is now more challenging with the identification of this new MßL.


Assuntos
Spirochaeta/enzimologia , Zinco/metabolismo , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Filogenia , Conformação Proteica , Zinco/química , beta-Lactamases/química , beta-Lactamas/química
14.
Elife ; 72018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30028291

RESUMO

Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the 'resting' and 'activated' structures suggest a mechanism for ligand discrimination.


Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Spirochaeta/enzimologia
15.
J Gen Physiol ; 150(6): 821-834, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29752414

RESUMO

Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.


Assuntos
Proteínas de Bactérias/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico , Animais , Proteínas de Bactérias/química , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Spirochaeta , Xenopus
16.
PLoS One ; 12(8): e0182280, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28771537

RESUMO

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.


Assuntos
Metagenômica , Pinctada/genética , Spirochaeta/patogenicidade , Exoesqueleto/microbiologia , Animais , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Hemolinfa/microbiologia , Hibridização in Situ Fluorescente , Penicilinas/farmacologia , Filogenia , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/isolamento & purificação , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Spirochaeta/classificação , Spirochaeta/efeitos dos fármacos , Infecções por Spirochaetales/genética , Infecções por Spirochaetales/patologia , Infecções por Spirochaetales/veterinária
17.
J Biol Chem ; 292(12): 4847-4860, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28179427

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Spirochaeta/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Metabolismo dos Carboidratos , Parede Celular/metabolismo , Celulases/química , Celulose/metabolismo , Cristalografia por Raios X , Glucanos/metabolismo , Modelos Moleculares , Concentração Osmolar , Ligação Proteica , Conformação Proteica , Spirochaeta/química , Temperatura , Xilanos/metabolismo
18.
Int J Syst Evol Microbiol ; 67(5): 1288-1295, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28100313

RESUMO

The anaerobic, non-motile strain HMT was isolated from the naphthalene-degrading, sulfate-reducing enrichment culture N47. For 20 years, strain HMT has been a stable member of culture N47 although it is neither able to degrade naphthalene nor able to reduce sulfate in pure culture. The highest similarity of the 16S rRNA gene sequence of strain HMT (89 %) is with a cultivated member of the family Spirochaetaceae, Treponema caldariumstrain H1T (=DSM 7334T), an obligately anaerobic, thermophilic spirochaete isolated from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA. In contrast to this strain and the majority of spirochaete species described, strain HMT showed a rod-shaped morphology. Growth occurred at temperatures between 12 and 50 °C (optimum 37 °C) but the isolate was not able to grow at 60 °C. The strain fermented various sugars including d-glucose, d-fructose, lactose and sucrose. Addition of 0.1 % (w/v) yeast extract or 0.1 % (w/v) tryptone to the culture medium was essential for growth and could not be replaced by either the vitamin solutions tested or by 0.1 % (w/v) peptone or 0.1 % (w/v) casamino acids. The DNA G+C content of the isolate was 51.5 mol%. The major fatty acids were C14 : 0, C18 : 1ω13c, C16 : 1ω9t, C16 : 1ω11c and C16 : 1ω9c. Based on the unique morphology and the phylogenetic distance from the closest cultivated relative, a novel genus and species, Rectinema cohabitans gen. nov., sp. nov., is proposed. The type strain is strain HMT (=DSM 100378T=JCM 30982T).


Assuntos
Fontes Termais/microbiologia , Filogenia , Spirochaeta/classificação , Aminoácidos/química , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Oregon , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Spirochaeta/genética , Spirochaeta/isolamento & purificação , Spirochaetales/genética
19.
Appl Environ Microbiol ; 83(4)2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27940544

RESUMO

This study aimed to identify the differences in the oral microbial communities in saliva from patients with and without caries by performing sequencing with the Illumina MiSeq platform, as well as to further assess their relationships with environmental factors (salivary pH and iron concentration). Forty-three volunteers were selected, including 21 subjects with and 22 without caries, from one village in Gansu, China. Based on 966,255 trimmed sequences and clustering at the 97% similarity level, 1,303 species-level operational taxonomic units were generated. The sequencing data for the two groups revealed that (i) particular distribution patterns (synergistic effects or competition) existed in the subjects with and without caries at both the genus and species levels and (ii) both the salivary pH and iron concentration had significant influences on the microbial community structure. IMPORTANCE: The significant influences of the oral environment observed in this study increase the current understanding of the salivary microbiome in caries. These results will be useful for expanding research directions and for improving disease diagnosis, prognosis, and therapy.


Assuntos
Ferro/análise , Microbiota/genética , Boca/microbiologia , Saliva/microbiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Adulto , Bacteroidetes/classificação , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Sequência de Bases , DNA Bacteriano/genética , Cárie Dentária/microbiologia , Firmicutes/classificação , Firmicutes/genética , Firmicutes/isolamento & purificação , Fusobactérias/classificação , Fusobactérias/genética , Fusobactérias/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Pessoa de Meia-Idade , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Análise de Sequência de DNA , Spirochaeta/classificação , Spirochaeta/genética , Spirochaeta/isolamento & purificação
20.
Int J Syst Evol Microbiol ; 66(12): 5485-5492, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27902269

RESUMO

Strain JC231 was isolated from a coastal saline habitat of Gujarat and was identified based on 16S rRNA gene sequence analysis as a member belonging to the genus Spirochaeta and showed highest sequence similarity (<91 %) with Spirochaeta bajacaliforniensis DSM 16054T and other members of the family Spirochaetaceae. Intensive attempts to culture strain JC231 in pure culture have failed and were associated with only one species of a Desulfovibrio. However, presence of fosmidomycin inhibited the growth of Desulfovibrio sp. and strain JC231 was characterized in its presence. Strain JC231 was an obligate anaerobe, helical shaped and Gram-stain-negative with catalase and oxidase negative. Draft genome sequence analysis of strain JC231 indicated the full complement of genes for both 2-C-methyl-d-erythritol 4-phosphate and 3-hydroxy-3-methylglutaryl-CoA pathways of terpenogenesis. C14 : 0, iso-C15 : 0, C16 : 0, iso-C15 : 1H/C13 : 0 3OH and iso-C14 : 0 are the major (>5 %) fatty acids. Strain JC231 contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and six unidentified lipids (L1-L6). G+C content of strain JC231 was 55.7 mol%. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain JC231 as a representative of a new genus and species in the family Spirochaetaceae, for which the name 'CandidatusMarispirochaeta associata' is proposed. Strain JC231 is deposited as a defined co-culture with Desulfovibrio sp. JC271 to DSMZ (DSM 29857) and KCTC (KCTC 15472). Based on phenotypic, genotypic and phylogenetic analyses, we also propose the reclassification of Spirochaeta bajacaliforniensis as Sediminispirochaeta bajacaliforniensis gen. nov., comb. nov., Spirochaeta smaragdinae as Sediminispirochaeta smaragdinae comb. nov. and Spirochaeta sinaica as Sediminispirochaeta sinaica comb. nov.


Assuntos
Filogenia , Spirochaeta/classificação , Acil Coenzima A/genética , Técnicas de Tipagem Bacteriana , Técnicas de Cocultura , DNA Bacteriano/genética , Eritritol/análogos & derivados , Eritritol/genética , Ácidos Graxos/química , Índia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Fosfatos Açúcares/genética
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