Enzyme-Primed Native Chemical Ligation Produces Autoinducing Cyclopeptides in Clostridia.

Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures remain elusive. Here, we report the genome-guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis.

IMPROvER: the Integral Membrane Protein Stability Selector.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .

Structures of the stator complex that drives rotation of the bacterial flagellum.

The bacterial flagellum is the prototypical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex. The motor consists of a central rotor surrounded by stator units that couple ion flow across the cytoplasmic membrane to generate torque. Here, we present the structures of the stator complexes from Clostridium sporogenes, Bacillus subtilis and Vibrio mimicus, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly where the five A subunits enclose the two B subunits. Comparison to structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion flow to generate mechanical work at a distance and suggests that such events involve rotation in the motor structures.

Characterization of a novel Clostridium sp. SP17-B1 and its application for succinic acid production from hevea wood waste hydrolysate.

An anaerobic, gram-positive, rod-shaped bacterium strain SP17-B1, isolated from dog saliva, was taxonomically characterized on the basis of phenotypic, chemotaxonomic, and genotypic characteristics. It was cultured in 4% (w/v) NaCl at a pH range of 5.0-8.0 (optimally at pH 7) and at 30°C-40 °C (optimally at 37 °C). Its major cellular fatty acids are C16:0 (36.3%), C17:0 cyclo (9.7%), C16:1ω9c (13.9%), and C18:1ω9c (10.7%), and its DNA guanine-cytosine content is 40.8 mol%. On the basis of the 16S rRNA gene sequence analysis, it was determined that the strain belonged to the genus Clostridium and was closely related to C. amygdalinum BR-10T (97.8%), C. saccharolyticum WM1T (97.8%), and C. celleracrescens DSM 5628T (97.7%). This strain showed a low level of DNA-DNA relatedness with the closely related strains, suggesting that it is a novel species in the genus Clostridium. Recent studies have demonstrated the production of succinic acid using Clostridium strains. Strain SP17-B1 produced 25.1 ±â€¯1.3 and 15.3 ±â€¯1.5 g/L of succinic acid from 40 g/L of glucose and 30 g/L of hevea wood waste hydrolysate (HH), respectively, after 24 h. When detoxified HH was used as a substrate, the lag phase was reduced and cell growth was enhanced by 7 fold (OD660 0.4-3.0) within 12 h. Detoxification using granular activated carbon may have reduced the levels of furfural and HMF without interfering with the amount of sugars in HH.

Application of MALDI-TOF MS to rapid identification of anaerobic bacteria.

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been rapidly developed and widely used as an analytical technique in clinical laboratories with high accuracy in microorganism identification. OBJECTIVE: To validate the efficacy of MALDI-TOF MS in identification of clinical pathogenic anaerobes. METHODS: Twenty-eight studies covering 6685 strains of anaerobic bacteria were included in this meta-analysis. Fixed-effects models based on the P-value and the I-squared were used for meta-analysis to consider the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 12.0. RESULTS: The identification accuracy of MALDI-TOF MS was 84% for species (I2 = 98.0%, P < 0.1), and 92% for genus (I2 = 96.6%, P < 0.1). Thereinto, the identification accuracy of Bacteroides was the highest at 96% with a 95% CI of 95-97%, followed by Lactobacillus spp., Parabacteroides spp., Clostridium spp., Propionibacterium spp., Prevotella spp., Veillonella spp. and Peptostreptococcus spp., and their correct identification rates were all above 90%, while the accuracy of rare anaerobic bacteria was relatively low. Meanwhile, the overall capabilities of two MALDI-TOF MS systems were different. The identification accuracy rate was 90% for VITEK MS vs. 86% for MALDI biotyper system. CONCLUSIONS: Our research showed that MALDI-TOF-MS was satisfactory in genus identification of clinical pathogenic anaerobic bacteria. However, this method still suffers from different drawbacks in precise identification of rare anaerobe and species levels of common anaerobic bacteria.

Anaerobic reduction of europium by a Clostridium strain as a strategy for rare earth biorecovery.

The biorecovery of europium (Eu) from primary (mineral deposits) and secondary (mining wastes) resources is of interest due to its remarkable luminescence properties, important for modern technological applications. In this study, we explored the tolerance levels, reduction and intracellular bioaccumulation of Eu by a site-specific bacterium, Clostridium sp. 2611 isolated from Phalaborwa carbonatite complex. Clostridium sp. 2611 was able to grow in minimal medium containing 0.5 mM Eu3+. SEM-EDX analysis confirmed an association between Eu precipitates and the bacterium, while TEM-EDX analysis indicated intracellular accumulation of Eu. According to the HR-XPS analysis, the bacterium was able to reduce Eu3+ to Eu2+ under growth and non-growth conditions. Preliminary protein characterization seems to indicate that a cytoplasmic pyruvate oxidoreductase is responsible for Eu bioreduction. These findings suggest the bioreduction of Eu3+ by Clostridium sp. as a resistance mechanism, can be exploited for the biorecovery of this metal.

Lipidomic Analysis of Clostridium cadaveris and Clostridium fallax.

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin-layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk-1-enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd2 Gro), which is present in tetraacyl, monoalkenyl-triacyl, and dialkenyl-diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine-phosphate-modified diacylglycerol. The significance of these lipid compositions is discussed.

Biosynthesis of Diverse Antimicrobial and Antiproliferative Acyloins in Anaerobic Bacteria.

Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)-dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum.

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σI ) factors that have cognate membrane-associated anti-σI factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σI4 -dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σI3 -dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14A ) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

The Polyextremophilic Bacterium Clostridium paradoxum Attains Piezophilic Traits by Modulating Its Energy Metabolism and Cell Membrane Composition.

In polyextremophiles, i.e., microorganisms growing preferentially under multiple extremes, synergistic effects may allow growth when application of the same extremes alone would not. High hydrostatic pressure (HP) is rarely considered in studies of polyextremophiles, and its role in potentially enhancing tolerance to other extremes remains unclear. Here, we investigated the HP-temperature response in Clostridium paradoxum, a haloalkaliphilic moderately thermophilic endospore-forming bacterium, in the range of 50 to 70°C and 0.1 to 30 MPa. At ambient pressure, growth limits were extended from the previously reported 63°C to 70°C, defining C. paradoxum as an actual thermophile. Concomitant application of high HP and temperature compared to standard conditions (i.e., ambient pressure and 50°C) remarkably enhanced growth, with an optimum growth rate observed at 22 MPa and 60°C. HP distinctively defined C. paradoxum physiology, as at 22 MPa biomass, production increased by 75% and the release of fermentation products per cell decreased by >50% compared to ambient pressure. This metabolic modulation was apparently linked to an energy-preserving mechanism triggered by HP, involving a shift toward pyruvate as the preferred energy and carbon source. High HPs decreased cell damage, as determined by Syto9 and propidium iodide staining, despite no organic solute being accumulated intracellularly. A distinct reduction in carbon chain length of phospholipid fatty acids (PLFAs) and an increase in the amount of branched-chain PLFAs occurred at high HP. Our results describe a multifaceted, cause-and-effect relationship between HP and cell metabolism, stressing the importance of applying HP to define the boundaries for life under polyextreme conditions.IMPORTANCE Hydrostatic pressure (HP) is a fundamental parameter influencing biochemical reactions and cell physiology; however, it is less frequently applied than other factors, such as pH, temperature, and salinity, when studying polyextremophilic microorganisms. In particular, how HP affects microbial tolerance to other and multiple extremes remains unclear. Here, we show that under polyextreme conditions of high pH and temperature, Clostridium paradoxum demonstrates a moderately piezophilic nature as cultures grow to highest cell densities and most efficiently at a specific combination of temperature and HP. Our results highlight the importance of considering HP when exploring microbial physiology under extreme conditions and thus have implications for defining the limits for microbial life in nature and for optimizing industrial bioprocesses occurring under multiple extremes.

Anaerobic Production of Poly(3-hydroxybutyrate) and Its Precursor 3-Hydroxybutyrate from Synthesis Gas by Autotrophic Clostridia.

Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.

Effects of fermentation products of the commensal bacterium Clostridium ramosum on motility, intracellular pH, and flagellar synthesis of enterohemorrhagic Escherichia coli.

The flagellum and motility are crucial virulence factors for many pathogenic bacteria. In general, pathogens invade and translocate through motility and adhere to specific tissue via flagella. Therefore, the motility and flagella of pathogens are effectual targets for attenuation. Here, we show that the fermentation products of Clostridium ramosum, a commensal intestinal bacterium, decrease the intracellular pH of enterohemorrhagic Escherichia coli (EHEC) and influence its swimming motility. Quantifications of flagellar rotation in individual EHEC cells showed an increase in reversal frequency and a decrease in rotation rate in the presence of C. ramosum fermentation products. Furthermore, the C. ramosum fermentation products affected synthesis of flagellar filaments. The results were reproduced by a combination of organic acids under acidic conditions. Short-chain fatty acids produced by microbes in the gut flora are beneficial for the host, e.g. they prevent infection. Thus, C. ramosum could affect the physiologies of other enteric microbes and host tissues.

Epidemiología de bacterias anaerobias aisladas en muestras clínicas en el hospital san juan de dios, San José, Costa Rica, durante el trienio 2014, 2015 y 2016 / Epidemiology of anaerobic bacteria isolated in clinical samples at San Juan de Dios Hospital, San José, Costa Rica, during the 2014, 2015 and 2016 triennium

Resumen La epidemiología de las posibles poblaciones en riesgo de sufrir una infección por bacterias anaerobias a nivel nacional es desconocida, se debería de incentivar el conocimiento en los servicios de salud sobre este tipo de infecciones. Las bacterias anaerobias están relacionadas en los medios hospitalarios como causa importante de morbilidad, razón por la cual es conveniente conocer la epidemiologia y prevalecía de especies involucradas. En el Laboratorio de Bacteriología del Hospital San Juan de Dios, durante tres años, se analizaron un total de 1545 pacientes sospechosos de microorganismos anaerobios en medios prerreducidos, mediante un tamizaje se redujeron a un total de 469 posibles muestras, las cuales, fueron enviadas al Laboratorio de Investigación en Bacteriología Anaerobia (LIBA) para su correspondiente identificación. A lo largo de las semanas epidemiológicas de los tres años se encontraron en promedio de 1.77 casos confirmados / semana, con razón de sexo positiva a favor de los masculinos. Se determinó que solo 245 de las muestras enviadas presentaban uno o varios microorganismos anaerobios estrictos representando un 15.85% del total, identificándose 39 especies diferentes, en 306 cepas aisladas. Las mayormente importante fue el género Bacteriodes, provenientes de cavidad abdominal seguido de abscesos y heridas de piel. El presente estudio tiene como objetivo presentar datos que respalden la importancia clínica de la búsqueda de microorganismos anaerobios y que ayuden a los analistas de bacteriología a guiar cuales son los principales microorganismos esperables en muestras clínicas, además de conocer la prevalencia en general.

Abstract The epidemiology of the possible populations at risk of suffering an infection by anaerobic bacteria a national level is unknown, it should be encouraged the knowledge in the health services about this type of infections. Anaerobic bacteria are related in hospital environments as an important cause of morbidity, which is why it is convenient to know the epidemiology and prevalence of species involved. In the Bacteriology Laboratory of the Hospital San Juan de Dios, for three years, a total of 1545 patients suspected of anaerobic microorganisms in prereduced media were analyzed, through a screening was reduced to a total of 469 possible samples, which were sent to the Anaerobic Bacteriology Research Laboratory (LIBA) for its corresponding identification. Throughout the epidemiological weeks of the three years were found on average of 1.77 confirmed cases / week, with a positive sex ratio in favor of men. It was determined that only 245 of the samples sent had one or several strict anaerobic microorganisms representing 15.85% of the total, identifying 39 different species, in 306 isolated strains. The most important was the genus Bacteriodes, coming from the abdominal cavity followed by abscesses and skin wounds. The present study aims to present data that support the clinical importance of the search for anaerobic microorganisms and that help the analysts of bacteriology to guide which are the main expected microorganisms in clinical samples, in addition to knowing the prevalence in general.

Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.

Bottom-up proteomics is increasingly being used to characterize unknown environmental, clinical, and forensic samples. Proteomics-based bacterial identification typically proceeds by tabulating peptide "hits" (i.e., confidently identified peptides) associated with the organisms in a database; those organisms with enough hits are declared present in the sample. This approach has proven to be successful in laboratory studies; however, important research gaps remain. First, the common-practice reliance on unique peptides for identification is susceptible to a phenomenon known as signal erosion. Second, no general guidelines are available for determining how many hits are needed to make a confident identification. These gaps inhibit the transition of this approach to real-world forensic samples where conditions vary and large databases may be needed. In this work, we propose statistical criteria that overcome the problem of signal erosion and can be applied regardless of the sample quality or data analysis pipeline. These criteria are straightforward, producing a p-value on the result of an organism or toxin identification. We test the proposed criteria on 919 LC-MS/MS data sets originating from 2 toxins and 32 bacterial strains acquired using multiple data collection platforms. Results reveal a > 95% correct species-level identification rate, demonstrating the effectiveness and robustness of proteomics-based organism/toxin identification.

Discrimination of Human Pathogen Clostridium Species Especially of the Heterogeneous C. sporogenes and C. botulinum by MALDI-TOF Mass Spectrometry.

Clostridium species cause several local and systemic diseases. Conventional identification of these microorganisms is in part laborious, not always reliable, time consuming or does not always distinguish different species, i.e., C. botulinum and C. sporogenes. All in, there is a high interest to find out a reliable, powerful and rapid method to identify Clostridium spp. not only on genus but also on species level. The aim of the present study was to identify Clostridium spp. strains and also to find differences and metabolic groups of C. botulinum by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). A total of 123 strains of Clostridium spp. (C. botulinum, n = 40, C. difficile, n = 11, C. tetani, n = 11, C. sordellii, n = 20, C. sporogenes, n = 18, C. innocuum, n = 10, C. perfringens, n = 13) were analyzed by MALDI-TOF MS in combination with methods of multivariate statistical analysis. MALDI-TOF MS analysis in combination with methods of multivariate statistical analysis was able to discriminate between the different tested Clostridium spp., even between species which are closely related and difficult to differentiate by traditional methods, i.e., C. sporogenes and C. botulinum. Furthermore, the method was able to separate the different metabolic groups of C. botulinum. Especially, E gene-positive C. botulinum strains are clearly distinguishable from the other species but also from those producing other toxin types. Thus, MALDI-TOF MS represents a reliable and above all quick method for identification of cultivated Clostridium species.

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii.

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.

Identification of Clostridium species using the VITEK® MS.

The genus Clostridium is of high clinical relevance, as some species may cause rapid and even lethal infections. Thus, a timely identification of these anaerobic bacteria is desirable. Conventional identification methods rely on biochemical properties of these organisms, however, establishing these is time-consuming and not always reliable. Alternatively, 16S rRNA gene sequence based diagnostic methods may be used, but they are expensive and not ubiquitously available. This study was designed to assess the possibility to identify Clostridium species employing the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, 848 Clostridium strains representing 42 species were analyzed with the VITEK® MS instrument (bioMérieux, Marcy l'Etoile, France), comparing mass spectra derived from these organisms with the spectra provided in the available database. 90.3% of the strains were correctly identified at species level and another 3.6% at genus level. Since the number of Clostridium species included in the database was rather limited (21 altogether), the spectra obtained were also analyzed employing the Shimadzu Pro Series software. Thus, it became possible to create a dendrogram of the species included in this study.

PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01-0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6-14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.

Insights into the Mechanisms by Which Clostridial Neurotoxins Discriminate between Gangliosides.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the causative agents of the paralytic diseases botulism and tetanus, respectively. Entry of toxins into neurons is mediated through initial interactions with gangliosides, followed by binding to a protein co-receptor. Herein, we aimed to understand the mechanism through which individual neurotoxins recognize the carbohydrate motif of gangliosides. Using cell-based and in vitro binding assays, in conjunction with structure-driven site-directed mutagenesis, a conserved hydrophobic residue within the BoNTs that contributes to both affinity and specificity toward Sia5-containing gangliosides was identified. We demonstrate that targeted mutations within the Sia5 binding pocket result in the generation of neurotoxins that either bind and enter cells more efficiently (BoNT/A1 and BoNT/B) or display altered ganglioside binding specificity (TeNT). These data support a model in which recognition of Sia5 is largely driven by hydrophobic interactions between the sugar and the Sia5 binding site.

Quantifying CBM Carbohydrate Interactions Using Microscale Thermophoresis.

MicroScale Thermophoresis (MST) is an emerging technology for studying a broad range of biomolecular interactions with high sensitivity. The affinity constant can be obtained for a wide range of molecules within minutes based on reactions in microliters. Here, we describe the application of MST in quantifying two CBM-carbohydrate interactions, a CBM3a toward cellulose nanocrystals and a CBM4 against xylohexaose.