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We report on the characterization of a novel genomic assembly (ARYD3) recovered from formation water (17.6% salinity) and crude oil enrichment amended by isolated soy proteins (0.2%), and incubated for 100 days under anaerobic conditions at 50°C. Phylogenetic and phylogenomic analysis demonstrated that the ARYD3 is unaffiliated with all currently described bacterial phyla and candidate phyla, as evident by the low AAI (34.7%), shared gene content (19.4%), and 78.9% 16S rRNA gene sequence similarity to Halothiobacillus neapolitanus, its closest cultured relative. Genomic characterization predicts a slow-growing, non-spore forming, and non-motile Gram-negative rod. Adaptation to high salinity is potentially mediated by the production of the compatible solutes cyclic 2,3-diphosphoglycerate (cDPG), α-glucosylglycerate, as well as the uptake of glycine betaine. Metabolically, the genome encodes primarily aminolytic capabilities for a wide range of amino acids and peptides. Interestingly, evidence of propionate degradation to succinate via methyl-malonyl CoA was identified, suggesting possible capability for syntrophic propionate degradation. Analysis of ARYD3 global distribution patterns identified its occurrence in a very small fraction of Earth Microbiome Project datasets examined (318/27,068), where it consistently represented an extremely rare fraction (maximum 0.28%, average 0.004%) of the overall community. We propose the Candidatus name Mcinerneyibacterium aminivorans gen. nov, sp. nov. for ARYD3 T , with the genome serving as the type material for the novel family Mcinerneyibacteriaceae fam. nov., order Mcinerneyibacteriales ord. nov., class Mcinerneyibacteria class nov., and phylum Mcinerneyibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number VSIX00000000., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.

A metabolic, genomic and proteomic assessment of Fervidobacterium pennivorans strains was undertaken to clarify the metabolic and genetic capabilities of this Thermotogales species. The type strain Ven5 originally isolated from a hot mud spa in Italy, and a newly isolated strain (DYC) from a hot spring at Ngatamariki, New Zealand, were compared for metabolic and genomic differences. The fermentation profiles of both strains on cellobiose generated similar major end products (acetate, alanine, glutamate, H 2 , and CO 2 ). The vast majority of end products produced were redox neutral, and carbon balances were in the range of 95-115%. Each strain showed distinct fermentation profiles on sugar substrates. The genome of strain DYC was sequenced and shown to have high sequence similarity and synteny with F. pennivorans Ven5 genome, suggesting they are the same species. The unique genome regions in Ven5, corresponded to genes involved in the Entner-Doudoroff pathway confirming our observation of DYC's inability to utilize gluconate. Genome analysis was able to elucidate pathways involved in production of the observed end-products with the exception of alanine and glutamate synthesis which were resolved with less clarity due to poor sequence identity and missing critical enzymes within the pathway, respectively.

The phylum Thermotogae gathers thermophilic, hyperthermophic, mesophilic, and thermo-acidophilic anaerobic bacteria that are mostly originated from geothermally heated environments. The metabolic and phenotypic properties harbored by the Thermotogae species questions the evolutionary events driving the emergence of this early branch of the universal tree of life. Recent reshaping of the Thermotogae taxonomy has led to the description of a new genus, Pseudothermotoga, a sister group of the genus Thermotoga within the order Thermotogales. Comparative genomics of both Pseudothermotoga and Thermotoga spp., including 16S-rRNA-based phylogenetic, pan-genomic analysis as well as signature indel conservation, provided evidence that Thermotoga caldifontis and Thermotoga profunda species should be reclassified within the genus Pseudothermotoga and renamed as Pseudothermotoga caldifontis comb. nov. (type strain=AZM44c09 T ) and Pseudothermotoga profunda comb. nov. (type strain=AZM34c06 T ), respectively. In addition, based upon whole-genome relatedness indices and DNA-DNA Hybridization results, the reclassification of Pseudothermotoga lettingae and Pseudothermotoga subterranea as latter heterotypic synonyms of Pseudothermotoga elfii is proposed. Finally, potential genetic elements resulting from the distinct evolutionary story of the Thermotoga and Pseudothermotoga clades are discussed., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Temperature is one of the defining parameters of an ecological niche. Most organisms thrive within a temperature range that rarely exceeds ~30 °C, but the deep subsurface bacterium Kosmotoga olearia can grow over a temperature range of 59 °C (20-79 °C). To identify genes correlated with this flexible phenotype, we compared transcriptomes of K. olearia cultures grown at its optimal 65 °C to those at 30, 40, and 77 °C. The temperature treatments affected expression of 573 of 2224 K. olearia genes. Notably, this transcriptional response elicits re-modeling of the cellular membrane and changes in metabolism, with increased expression of genes involved in energy and carbohydrate metabolism at high temperatures and up-regulation of amino acid metabolism at lower temperatures. At sub-optimal temperatures, many transcriptional changes were similar to those observed in mesophilic bacteria at physiologically low temperatures, including up-regulation of typical cold stress genes and ribosomal proteins. Comparative genomic analysis of additional Thermotogae genomes indicates that one of K. olearia's strategies for low-temperature growth is increased copy number of some typical cold response genes through duplication and/or lateral acquisition. At 77 °C one-third of the up-regulated genes are of hypothetical function, indicating that many features of high-temperature growth are unknown.

To fully unlock the biotechnological potentials of Thermotoga species, this study aimed to expand the genetic toolbox of Thermotoga by developing a new selective system. The developed system was composed of two components: a recipient strain bearing a deletion in its orotate phosphoribosyltransferase gene pyrE and a shuttle vector expressing a heterologous pyrE as the selectable marker. A spontaneous uracil auxotroph, T. sp. strain RQ7-15, was isolated at 70 °C with 2 mg/ml 5-fluoroorotic acid. The mutant carried a 112 bp deletion in pyrE and was a suitable recipient strain. To avoid homologous recombination, the pyrE gene from another thermophilic bacterium Caldicellulosiruptor saccharolyticus was used as the selectable marker. The gene was cloned into two Thermotoga-E. coli shuttle vectors, controlled by different promoters: the promoter of Thermus S-layer protein (P slpA ) in pDH25 and the promoter of the pyrimidine synthesis operon of T. sp. strain RQ7 (P RQ7.pyr ) in pDH28. After being introduced into the mutant strain RQ7-15 through natural transformation, both vectors allowed the host to thrive in a minimal medium. Single colonies of transformants were isolated and confirmed by polymerase chain reactions and restriction digestions. In summary, a pyrE-based selective system has been established in T. sp. strain RQ7.

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol L329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

D-galacturonic acid is a potential platform chemical comprising the principal component of pectin in the citrus processing waste stream. Several enzyme activities are required for the enzymatic production of galacturonic acid from pectin, including exo- and endo-polygalacturonases. The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in Escherichia coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and details of their catalytic mechanism have been elucidated shows that three catalytic Asp residues and several other key active site residues are strictly conserved. Purified TtGH28 was dimeric and hyperthermostable, with K t (0.5)  = 86.3 °C. Kinetic parameters for activity on digalacturonic acid, trigalacturonic acid, and polygalacturonic acid were obtained. No substrate inhibition was observed for polygalacturonate, while the K si values for the oligogalacturonides were in the low mM range, and K i for product galacturonic acid was in the low μM range. Kinetic modeling of the progress of reaction showed that the enzyme is both fully exo- and fully non-processional.

Plant cell walls are composed of complex polysaccharides such as cellulose and hemicellulose. In order to efficiently hydrolyze cellulose, the synergistic action of several cellulases is required. Some anaerobic cellulolytic bacteria form multienzyme complexes, namely cellulosomes, while other microorganisms produce a portfolio of diverse enzymes that work in synergistic fashion. Molecular biological methods can mimic such effects through the generation of artificial bi- or multifunctional fusion enzymes. Endoglucanase and β-glucosidase from extremely thermophilic anaerobic bacteria Fervidobacterium gondwanense and Fervidobacterium islandicum, respectively, were fused end-to-end in an approach to optimize polysaccharide degradation. Both enzymes are optimally active at 90 °C and pH 6.0-7.0 representing excellent candidates for fusion experiments. The direct linkage of both enzymes led to an increased activity toward the substrate specific for β-glucosidase, but to a decreased activity of endoglucanase. However, these enzyme chimeras were superior over 1:1 mixtures of individual enzymes, because combined activities resulted in a higher final product yield. Therefore, such fusion enzymes exhibit promising features for application in industrial bioethanol production processes.

Objective: Chronic inflammation has earlier been detected in ruptured intracranial aneurysms. A previous study detected both dental bacterial DNA and bacterial-driven inflammation in ruptured intracranial aneurysm walls. The aim of this study was to compare the presence of oral and pharyngeal bacterial DNA in ruptured and unruptured intracranial aneurysms. The hypothesis was that oral bacterial DNA findings would be more common and the amount of bacterial DNA would be higher in ruptured aneurysm walls than in unruptured aneurysm walls., Materials and Methods: A total of 70 ruptured (n = 42) and unruptured (n = 28) intracranial aneurysm specimens were obtained perioperatively in aneurysm clipping operations. Aneurysmal sac tissue was analysed using a real-time quantitative polymerase chain reaction to detect bacterial DNA from several oral species. Both histologically non-atherosclerotic healthy vessel wall obtained from cardiac by-pass operations (LITA) and arterial blood samples obtained from each aneurysm patient were used as control samples., Results: Bacterial DNA was detected in 49/70 (70%) of the specimens. A total of 29/42 (69%) of the ruptured and 20/28 (71%) of the unruptured aneurysm samples contained bacterial DNA of oral origin. Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively). There was no significant difference in the amount of bacterial DNA between the ruptured and unruptured samples., Conclusion: Dental bacterial DNA can be found using a quantitative polymerase chain reaction in both ruptured and unruptured aneurysm walls, suggesting that bacterial DNA plays a role in the pathogenesis of cerebral aneurysms in general, rather than only in ruptured aneurysms.

The thermostable β-glucosidase gene from Thermotoga petrophila DSM 13995 was cloned and overexpressed in Escherichia coli. The activity of the recombinant β-glucosidase was 21 U/mL in the LB medium. Recombinant β-glucosidase was purified, and its molecular weight was approximately 81 kDa. The optimal activity was at pH 5.0 and 90 °C, and the thermostability of the enzyme was improved by Ca(2+). The β-glucosidase had high selectivity for cleaving the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1, which produced the pharmacologically active minor ginsenoside 20(S)-Rg3. In a reaction at 90 °C and pH 5.0, 10 g/L of ginsenoside Rb1 was transformed into 6.93 g/L of Rg3 within 90 min, with a corresponding molar conversion of 97.9%, and Rg3 productivity of 4620 mg/L/h. This study is the first report of a GH3-family enzyme that used Ca(2+) to improve its thermostability, and it is the first report on the high substrate concentration bioconversion of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 by using thermostable β-glucosidase under high temperature.

A thermophilic, anaerobic, iron-reducing bacterium (strain DY22619T) was isolated from a sulfide sample collected from an East Pacific Ocean hydrothermal field at a depth of 2901 m. Cells were Gram-stain-negative, motile rods (2-10 µm in length, 0.5 µm in width) with multiple peritrichous flagella. The strain grew at 40-70 °C inclusive (optimum 60 °C), at pH 4.5-8.5 inclusive (optimum pH 7.0) and with sea salts concentrations of 1-10 % (w/v) (optimum 3 % sea salts) and NaCl concentrations of 1.5-5.0 % (w/v) (optimum 2.5 % NaCl). Under optimal growth conditions, the generation time was around 55 min. The isolate was an obligate chemoorganoheterotroph, utilizing complex organic compounds, amino acids, carbohydrates and organic acids including peptone, tryptone, beef extract, yeast extract, alanine, glutamate, methionine, threonine, fructose, mannose, galactose, glucose, palatinose, rhamnose, turanose, gentiobiose, xylose, sorbose, pyruvate, tartaric acid, α-ketobutyric acid, α-ketovaleric acid, galacturonic acid and glucosaminic acid. Strain DY22619T was strictly anaerobic and facultatively dependent on various forms of Fe(III) as an electron acceptor: insoluble forms and soluble forms. It did not reduce sulfite, sulfate, thiosulfate or nitrate. The genomic DNA G+C content was 29.0 mol%. Phylogenetic 16S rRNA gene sequence analyses revealed that the closest relative of strain DY22619T was Caloranaerobacter azorensis MV1087T, sharing 97.41 % 16S rRNA gene sequence similarity. On the basis of physiological distinctness and phylogenetic distance, the isolate is considered to represent a novel species of the genus Caloranaerobacter, for which the name Caloranaerobacterhttp://dx.doi.org/10.1601/nm.4081ferrireducens sp. nov. is proposed. The type strain is DY22619T ( = JCM 19467T = DSM 27799T = MCCC1A06455T).

A strictly anaerobic, mesophilic, iron-reducing bacterial strain, IRF19(T), was isolated from coal-contaminated soil in the Republic of Korea. IRF19(T) cells were straight, rod-shaped, Gram-staining-negative and motile by means of flagella. The optimum pH and temperature for their growth were determined to be pH 7.5-8.0 and 40 °C, while the optimum range was pH 6.5-10.0 and 20-45 °C, respectively. Strain IRF19(T) did not require NaCl for growth but it tolerated up to 2% (w/v). Growth was observed with yeast extract, D-glucose, D-fructose, D-ribose, D-mannitol, D-mannose, L-serine, L-alanine and L-isoleucine. Fe(III), elemental sulfur, thiosulfate and sulfate were used as electron acceptors. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain IRF19(T) is affiliated to the family Clostridiaceae and is most closely related to Salimesophilobacter vulgaris Zn2(T) (93.5% similarity), Geosporobacter subterraneus VNs68(T) (93.2%) and Thermotalea metallivorans B2-1(T) (92.3%). The major cellular fatty acids of strain IRF19(T) were C14 : 0, iso-C15 : 0 and C16 : 0, and the profile was distinct from those of the closely related species. The major respiratory quinone of strain IRF19(T) was menaquinone MK-5 (V-H2). The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and two unknown polar lipids. The G+C content of the genomic DNA of strain IRF19(T) was determined to be 37.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic results, strain IRF19(T) is considered to represent a novel species of a novel genus of the family Clostridiaceae , for which we propose the name Anaerosolibacter carboniphilus gen. nov., sp. nov., with the type strain IRF19(T) ( =KCTC 15396(T) =JCM 19988(T))., (© 2015 IUMS.)

A novel phospholipase B (TLPLB) from Thermotoga lettingae TMO has been cloned, functionally overexpressed in Escherichia coli and purified to homogeneity. Gas chromatography indicated that the enzyme could efficiently hydrolyze both the sn-1 and sn-2 ester bonds of 1-palmitoyl-2-oleoyl phosphatidylcholine as phospholipase B. TLPLB was optimally active at 70 °C and pH 5.5, respectively. Its thermostability is relatively high with a half-life of 240 min at 90 °C. TLPLB also displayed remarkable organic solvent tolerance and maintained approximately 91-161 % of its initial activity in 20 and 50 % (v/v) hydrophobic organic solvents after incubation for 168 h. Furthermore, TLPLB exhibited high degumming activity towards rapeseed, soybean, peanut and sunflower seed oils, where the phosphorus contents were decreased from 225.2, 189.3, 85.6 and 70.4 mg/kg to 4.9, 4.7, 3.2 and 2.2 mg/kg within 5 h, respectively. TLPLB could therefore be used for the degumming of vegetable oils.

Background: Despite the large volume of genome sequencing data produced by next-generation sequencing technologies and the highly sophisticated software dedicated to handling these types of data, gaps are commonly found in draft genome assemblies. The existence of gaps compromises our ability to take full advantage of the genome data. This study aims to identify a practical approach for biologists to complete their own genome assemblies using commonly available tools and resources., Results: A pipeline was developed to assemble complete genomes primarily from the next generation sequencing (NGS) data. The input of the pipeline is paired-end Illumina sequence reads, and the output is a high quality complete genome sequence. The pipeline alternates the employment of computational and biological methods in seven steps. It combines the strengths of de novo assembly, reference-based assembly, customized programming, public databases utilization, and wet lab experimentation. The application of the pipeline is demonstrated by the completion of a bacterial genome, Thermotoga sp. strain RQ7, a hydrogen-producing strain., Conclusions: The developed pipeline provides an example of effective integration of computational and biological principles. It highlights the complementary roles that in silico and wet lab methodologies play in bioinformatical studies. The constituting principles and methods are applicable to similar studies on both prokaryotic and eukaryotic genomes.

A novel obligately anaerobic, extremely thermophilic, organotrophic bacterium, strain Rift-s3(T), was isolated from a deep-sea sample containing Riftia pachyptila sheath from Guaymas Basin, Gulf of California. Cells of the novel isolate were rods, 0.3-0.8 µm in width and 1.5-10 µm in length, surrounded by a sheath-like structure (toga). Strain Rift-s3(T) grew at temperatures ranging from 44 to 75 °C, at pH 5.5 to 8.0, and with NaCl concentrations of 3 to 60 g l(-1). Under optimum conditions (65 °C, pH 6.0, NaCl 25 g l(-1)), the doubling time was 30 min. The isolate was able to ferment mono-, oligo- and polysaccharides including cellulose, chitin, xylan and pectin, and proteins including β-keratins, casein and gelatin. Acetate, hydrogen and carbon dioxide were the main products of glucose fermentation. The G+C content of the DNA was 30 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed the affiliation of strain Rift-s3(T) with the genus Thermosipho, with Thermosipho atlanticus Ob7(T) as the closest relative (96.5 % 16S rRNA gene sequence similarity). Based on the phylogenetic analysis and physiological properties of the novel isolate we propose a novel species of the genus Thermosipho, Thermosipho activus sp. nov., with Rift-s3(T) ( = DSM 26467(T) = VKM B-2803(T)) as the type strain., (© 2014 IUMS.)

Objective: Bacteria play an important role in the onset and perpetuation of intestinal inflammation in inflammatory bowel disease (IBD). Unlike in Crohn's disease (CD), in which dysbiosis has been better characterised, in ulcerative colitis (UC), only small cohorts have been studied and showed conflicting data. Therefore, we evaluated in a large cohort if the microbial signature described in CD is also present in UC, and if we could characterise predominant dysbiosis in UC. To assess the functional impact of dysbiosis, we quantified the bacterial metabolites., Design: The predominant microbiota from 127 UC patients and 87 age and sex-matched controls was analysed using denaturing gradient gel electrophoresis (DGGE) analysis. Differences were quantitatively validated using real-time PCR. Metabolites were quantified using gas chromatography-mass spectrometry., Results: Based on DGGE analysis, the microbial signature previously described in CD was not present in UC. Real-time PCR analysis revealed a lower abundance of Roseburia hominis (p<0.0001) and Faecalibacterium prausnitzii (p<0.0001) in UC patients compared to controls. Both species showed an inverse correlation with disease activity. Short-chain fatty acids (SCFA) were reduced in UC patients (p=0.014), but no direct correlation between SCFA and the identified bacteria was found., Conclusions: The composition of the fecal microbiota of UC patients differs from that of healthy individuals: we found a reduction in R hominis and F prausnitzii, both well-known butyrate-producing bacteria of the Firmicutes phylum. These results underscore the importance of dysbiosis in IBD but suggest that different bacterial species contribute to the pathogenesis of UC and CD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

A novel anaerobic succinate-producing bacterium, strain ZWB(T), was isolated from sludge collected from a biogas desulfurization bioreactor (Eerbeek, the Netherlands). Cells were non-spore-forming, motile, slightly curved rods (0.4-0.5 µm in diameter and 2-3 µm in length), and stained Gram-negative. The temperature range for growth was 25-40 °C, with an optimum at 37 °C. The pH range for growth was 7.0-9.0, with an optimum at pH 7.5. Strain ZWB(T) was able to ferment glycerol and several carbohydrates mainly to H2, succinate and acetate. Sulfur and fumarate could be used as electron acceptors by strain ZWB(T). The G+C content of the genomic DNA was 37.6 mol%. The most abundant fatty acids were iso-C14 : 0 and iso-C16 : 0 DMA. On the basis of 16S rRNA gene sequence similarity, strain ZWB(T) belongs to the family Ruminococcaceae and it is distantly related to Saccharofermentans acetigenes JCM 14006(T) (92.1%). Based on the physiological features and phylogenetic analysis, strain ZWB(T) represents a novel species of a new genus, for which the name Ercella succinigenes gen. nov., sp. nov. is proposed. The type strain of Ercella succinigenes is ZWB(T) ( = DSM 27333(T) = JCM 19283(T))., (© 2014 IUMS.)

A mesophilic, obligately anaerobic, lactate-, alcohol-, carbohydrate- and amino-acid- degrading bacterium, designated strain 7WAY-8-7(T), was isolated from an upflow anaerobic sludge blanket reactor treating high-strength organic wastewater from isomerized sugar production processes. Cells of strain 7WAY-8-7(T) were motile, curved rods (0.7-1.0×5.0-8.0 µm). Spore formation was not observed. The strain grew optimally at 37 °C (range for growth was 25-40 °C) and pH 7.0 (pH 6.0-7.5), and could grow fermentatively on yeast extract, glucose, ribose, xylose, malate, tryptone, pyruvate, fumarate, Casamino acids, serine and cysteine. The main end-products of glucose fermentation were acetate and hydrogen. In co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864(T), strain 7WAY-8-7(T) could utilize lactate, glycerol, ethanol, 1-propanol, 1-butanol, L-glutamate, alanine, leucine, isoleucine, valine, histidine, asparagine, glutamine, arginine, lysine, threonine, 2-oxoglutarate, aspartate and methionine. A Stickland reaction was not observed with some pairs of amino acids. Yeast extract was required for growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite and Fe (III) were not used as terminal electron acceptors. The G+C content of the genomic DNA was 61.4 mol%. 16S rRNA gene sequence analysis revealed that the isolate belongs to the uncultured environmental clone clade (called 'PD-UASB-13' in the Greengenes database) in the bacterial phylum Synergistetes, showing less than 90% sequence similarity with closely related described species such as Aminivibrio pyruvatiphilus and Aminobacterium colombiense (89.7% and 88.7%, respectively). The major cellular fatty acids were iso-C(13 : 0), iso-C(15 : 0), anteiso-C(15 : 0), C(18 : 1), C(19 : 1), C(20 : 1) and C(21 : 1). A novel genus and species, Lactivibrio alcoholicus gen. nov., sp. nov. is proposed to accommodate strain 7WAY-8-7(T) ( = JCM 17151(T) = DSM 24196(T) = CGMCC 1.5159(T))., (© 2014 IUMS.)

Two thermophilic, strictly anaerobic, Gram-negative bacteria, designated strains AZM34c06(T) and AZM44c09(T), were isolated from terrestrial hot springs in Japan. The optimum growth conditions for strain AZM34c06(T) were 60 °C, pH 7.4 and 0% additional NaCl, and those for strain AZM44c09(T) were 70 °C, pH 7.4 and 0% additional NaCl. Complete genome sequencing was performed for both strains, revealing genome sizes of 2.19 Mbp (AZM34c06(T)) and 2.01 Mbp (AZM44c09(T)). Phylogenetic analyses based on 16S rRNA gene sequences and the concatenated predicted amino acid sequences of 33 ribosomal proteins showed that both strains belonged to the genus Thermotoga. The closest relatives of strains AZM34c06(T) and AZM44c09(T) were the type strains of Thermotoga lettingae (96.0% similarity based on the 16S rRNA gene and 84.1% similarity based on ribosomal proteins) and Thermotoga hypogea (98.6 and 92.7% similarity), respectively. Using blast, the average nucleotide identity was 70.4-70.5% when comparing strain AZM34c06(T) and T. lettingae TMO(T) and 76.6% when comparing strain AZM44c09(T) and T. hypogea NBRC 106472(T). Both values are far below the 95% threshold value for species delineation. In view of these data, we propose the inclusion of the two isolates in the genus Thermotoga within two novel species, Thermotoga profunda sp. nov. (type strain AZM34c06(T) = NBRC 106115(T) = DSM 23275(T)) and Thermotoga caldifontis sp. nov. (type strain AZM44c09(T) = NBRC 106116(T) = DSM 23272(T))., (© 2014 IUMS.)

A gram-negative, motile, fermentative, thermophilic bacterium, designated AR80(T), was isolated from a high-temperature oil reservoir in Yabase Oilfield in Akita, Japan. Cells were rod-shaped, motile by means of polar flagella, and formed circular, convex, white colonies. The strain grew at 40-65 °C (optimum 60 °C), 0.5-9 % (w/v) NaCl (optimum 0.5-1 %), pH 6-9 (optimum pH 7.5), and elemental sulfur or thiosulfate serves as terminal electron acceptor. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain AR80(T) belonged to the genus Petrotoga and shared approximately 94.5 % sequence similarity with the type species of this genus. The G + C content of genomic DNA was 32.4 mol% while the value of DNA-DNA hybridization between the closest relative species Petrotoga miotherma and AR80(T) was 58.1 %. The major cellular fatty acids of strain AR80(T) consisted of 18:1 w9c, 16:0, and 16:1 w9c. Based on genetic and phenotypic properties, strain AR80(T) was different with other identified Petrotoga species and represents as a novel species, for which the name Petrotoga japonica sp. nov. is proposed. The type strain is AR80(T) (=NBRC 108752(T) = KCTC 15103(T) = HUT 8122(T)).

Background: Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated., Results: The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent K(m) and V(max) values were determined to be 12.3 ± 0.15 mg ml⁻¹ and 1,052.1 ± 12.7 μmol ml⁻¹ min⁻¹, respectively (at pH 6.5, 75°C), and the calculated kcat value was 349.3 ± 4.2 s⁻¹., Conclusions: This work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.

A taxonomic study employing a polyphasic approach was performed on a novel anaerobic bacterium isolated from natural gas production-water. The bacterium stained Gram-negative and consisted of non-motile, non-spore-forming, rod-shaped cells. Products of glucose or starch fermentation were ethanol, CO2, formate, acetate and H2. The predominant fatty acids were C16 : 0 ALDE and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c. The DNA G+C content was 45.5 mol%. 16S rRNA gene sequence analysis demonstrated that the nearest phylogenetic neighbours of the novel strain were Acetivibrio multivorans DSM 6139(T) (98.5 %) and Proteiniclasticum ruminis JCM 14817(T) (95.4 %). The DNA-DNA hybridization value between the novel organism and Acetivibrio multivorans PeC1 DSM 6139(T) was determined to be only 30.2 %, demonstrating the separateness of the two species. Based on phylogenetic, phenotypic and chemotaxonomic evidence that clearly distinguished strain 232.1(T) from Proteiniclasticum ruminis and other close relatives, it is proposed that the novel isolate be classified as representing a novel species of a new genus within the family Clostridiaceae, Youngiibacter fragilis gen. nov., sp. nov. The type strain of the type species is 232.1(T) ( = ATCC BAA-2257(T) = DSM 24749(T)). In addition, Acetivibrio multivorans is proposed to be reclassified as Youngiibacter multivorans comb. nov.

A novel moderately thermophilic, heterotrophic bacterium was isolated from a deep-sea hydrothermal vent deposit from the Mariner field along the Eastern Lau Spreading Center of the south-western Pacific Ocean. Cells were short motile rods (about 0.4×0.8 µm) that occurred singly or in pairs and were surrounded by a sheath-like membrane or 'toga'. The cells grew between 45 and 65 °C (optimum 57-60 °C) and at pH 4.1-6.0 (optimum pH 5.5-5.7) and grew optimally at 3 % (w/v) NaCl. The isolate grew on a range of carbon and proteinaceous substrates and reduced sulfur. The G+C content of the DNA was about 45 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed the new isolate as a deeply diverging lineage within the order Thermotogales. Based on the physiological, morphological and phylogenetic data, the isolate represents a novel species of a new genus with the proposed name Mesoaciditoga lauensis gen. nov., sp. nov. The type strain of Mesoaciditoga lauensis is cd-1655R(T) ( = DSM 25116(T) = OCM 1212(T)).

Optimal growth temperature is a complex trait involving many cellular components, and its physiology is not yet fully understood. Evolution of continuous characters, such as optimal growth temperature, is often modeled as a one-dimensional random walk, but such a model may be an oversimplification given the complex processes underlying the evolution of continuous characters. Recent articles have used ancestral sequence reconstruction to infer the optimal growth temperature of ancient organisms from the guanine and cytosine content of the stem regions of ribosomal RNA, allowing inferences about the evolution of optimal growth temperature. Here, we investigate the optimal growth temperature of the bacterial phylum Thermotogae. Ancestral sequence reconstruction using a nonhomogeneous model was used to reconstruct the stem guanine and cytosine content of 16S rRNA sequences. We compare this sequence reconstruction method with other ancestral character reconstruction methods, and show that sequence reconstruction generates smaller confidence intervals and different ancestral values than other reconstruction methods. Unbiased random walk simulation indicates that the lower temperature members of the Thermotogales have been under directional selection; however, when a simulation is performed that takes possible mutations into account, it is the high temperature lineages that are, in fact, under directional selection. We find that the evolution of Thermotogales optimal growth temperatures is best fit by a biased random walk model. These findings suggest that it may be easier to evolve from a high optimal growth temperature to a lower one than vice versa.

We recently reported that the Thermotogales acquired the ability to synthesize vitamin B12 by acquisition of genes from two distantly related lineages, Archaea and Firmicutes (K. S. Swithers et al., Genome Biol. Evol. 4:730-739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in the Thermotogales' most recent common ancestor. We also predicted that Thermotoga lettingae could not synthesize B12 de novo but could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found that Tt. lettingae did not synthesize B12 de novo but salvaged cobinamide. The growth rate of Tt. lettingae increased with the addition of B12 or cobinamide to its medium. It synthesized B12 when the medium was supplemented with cobinamide, and no B12 was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12 riboswitch. In other organisms, B12 riboswitches allow for higher transcriptional activity in the absence of B12. When Tt. lettingae was grown with no B12, the salvage genes were upregulated compared to cells grown with B12 or cobinamide. Another gene cluster with a putative B12 riboswitch upstream is the btuFCD ABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shown in vitro to bind both B12 and cobinamide. These results suggest that Thermotogales species can use the BtuFCD transporter to import both B12 and cobinamide, even if they cannot salvage cobinamide.

Background: Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above., Results: A β-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters K(m) and V(max) values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min⁻¹ mg⁻¹, 58.34±1.75 mg mL⁻¹ and 285.71±10.86 μmol min⁻¹ mg⁻¹, respectively. The k(cat)/K(m) values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM⁻¹ s⁻¹ and 41.47±1.58 s⁻¹ mg⁻¹ mL, respectively. It displayed high tolerance to mannose, with a K(i) value of approximately 900 mM., Conclusions: This work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.

We recently introduced a new molecular evolution model called the IDIS model for Insertion Deletion Independent of Substitution [13,14]. In the IDIS model, the three independent processes of substitution, insertion and deletion of residues have constant rates. In order to control the genome expansion during evolution, we generalize here the IDIS model by introducing an insertion rate which decreases when the sequence grows and tends to 0 for a maximum sequence length nmax. This new model, called LIIS for Limited Insertion Independent of Substitution, defines a matrix differential equation satisfied by a vector P(t) describing the sequence content in each residue at evolution time t. An analytical solution is obtained for any diagonalizable substitution matrix M. Thus, the LIIS model gives an expression of the sequence content vector P(t) in each residue under evolution time t as a function of the eigenvalues and the eigenvectors of matrix M, the residue insertion rate vector R, the total insertion rate r, the initial and maximum sequence lengths n0 and nmax, respectively, and the sequence content vector P(t0) at initial time t0. The derivation of the analytical solution is much more technical, compared to the IDIS model, as it involves Gauss hypergeometric functions. Several propositions of the LIIS model are derived: proof that the IDIS model is a particular case of the LIIS model when the maximum sequence length nmax tends to infinity, fixed point, time scale, time step and time inversion. Using a relation between the sequence length l and the evolution time t, an expression of the LIIS model as a function of the sequence length l=n(t) is obtained. Formulas for 'insertion only', i.e. when the substitution rates are all equal to 0, are derived at evolution time t and sequence length l. Analytical solutions of the LIIS model are explicitly derived, as a function of either evolution time t or sequence length l, for two classical substitution matrices: the 3-parameter symmetric substitution matrix [12] (LIIS-SYM3) and the HKY asymmetric substitution matrix[9] (LIIS-HKY). An evaluation of the LIIS model (precisely, LIIS-HKY) based on four statistical analyses of the GC content in complete genomes of four prokaryotic taxonomic groups, namely Chlamydiae, Crenarchaeota, Spirochaetes and Thermotogae, shows the expected improvement from the theory of the LIIS model compared to the IDIS model., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Strain VNs100(T), a novel mesophilic, anaerobic, rod-coccoid-shaped bacterium, having a sheath-like outer structure (toga), was isolated from a water sample collected in the area of an underground gas storage aquifer. It was non-motile with cells appearing singly (2-4 µm long × 1-2 µm wide), in pairs or as long chains and stained Gram-negative. Strain VNs100(T) was heterotrophic, able to use arabinose, cellobiose, fructose, galactose, glucose, lactose, lactate, mannose, maltose, raffinose, ribose, sucrose and xylose as energy sources only in the presence of elemental sulfur as terminal electron acceptor. Acetate, CO2 and sulfide were the end products of sugar metabolism. Hydrogen was not detected. Elemental sulfur, but not thiosulfate, sulfate or sulfite, were reduced to sulfide. Strain VNs100(T) grew at temperatures between 30 and 50 °C (optimum 45 °C), at pH values between 6.2 and 7.9 (optimum 7.3-7.5) and at NaCl concentrations between 0 and 15 g l(-1) (optimum 2 g l(-1)). The DNA G+C content was 47.5 mol%. The main cellular fatty acid was C16 : 0. Phylogenetic analysis of the small subunit rRNA gene sequence indicated that strain VNs100(T) had as its closest relatives 'Mesotoga sulfurireducens' (97.1 % similarity) and Mesotoga prima (similarity of 97.1 % and 97.7 % with each of its two genes, respectively) within the order Thermotogales. Hybridization between strain VNS100(T) and 'M. sulfurireducens' and between strain VNS100(T) and M. prima showed 12.9 % and 20.6 % relatedness, respectively. Based on phenotypic, phylogenetic and taxonomic characteristics, strain VNs100(T) is proposed as a representative of a novel species of the genus Mesotoga in the family Thermotogaceae, order Thermotogales. The name Mesotoga infera sp. nov. is proposed. The type strain is VNs100(T) (= DSM 25546(T) = JCM 18154(T)).

A novel anaerobic, mesophilic, amino-acid-fermenting bacterium, designated strain CL-84(T), was isolated from the swine intestinal tract on mucin-based media. Cells were curved rods (0.8-1.2 × 3.5-5.0 µm), stained Gram-negative and were non-motile with no evidence of spores. Strain CL-84(T) produced acetate, propionate, formate and butyrate as the end products of metabolism when grown on serine. Optimum growth occurred at 39 °C and pH 6.5. The major cellular fatty acids were iso-C15:0, iso-C15:0 3-OH, iso-C17:0 and C16:0, distinguishing strain CL-84(T) from closely related species. The DNA G+C content of strain CL-84(T) was 55.1 mol%. 16S rRNA gene sequence analysis showed that strain CL-84(T) shared 90-95% similarity with characterized genera within the phylum Synergistetes, family Synergistaceae. Phylogenetic analysis showed that strain CL-84(T) was related to, but distinct from, Cloacibacillus evryensis. Based on these findings, we propose that strain CL-84(T) represents a novel species of the genus Cloacibacillus. We further propose the name Cloacibacillus porcorum sp. nov. be designated for this species. The type strain is CL-84(T) (=DSM 25858(T)=CCUG 62631(T)). An emended description of the genus Cloacibacillus is provided.

Objective: Information about the spectrum of microorganisms in the intraimplant cavities of two-piece dental implants is scarce. The purpose of this study was to assess the intraimplant microflora of two-piece dental implants by conventional biochemical testing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16 s rDNA gene sequencing., Materials and Methods: Ten patients (six men and four women; average age = 66.7 years; age range = 58-78 years) received 35 two-piece titanium implants carrying ball attachments. Biofilm sampling was performed with sterile microbrushes, and nonadherent microbial samples were obtained by injection and reuptake of predefined volumes of NaCl solution. The samples were cultured and analyzed by conventional biochemical testing, MALDI-TOF MS, and 16 s rDNA gene sequencing., Results: Of the 103 species detected, 27 and 33 were identified only in the biofilm and nonadherent microbial samples, respectively. Forty-three species were identified in both types of samples., Conclusions: Two-piece dental implants harbored a broad spectrum of gram-positive and gram-negative aerobes and anaerobes, especially rods and cocci., Clinical Relevance: These findings confirm bacterial translocation from the oral cavity to intraimplant cavities. Microbiological methods as used in this study are necessary to reveal the complete vital microflora of intraimplant cavities.

The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted D-psicose to D-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k m, turnover number (k cat), and catalytic efficiency (k cat k m (-1) ) for substrate D-psicose were 64 mM, 6.98 min(-1) and 0.11 mM(-1) min(-1) respectively.

A moderately thermophilic and strictly anaerobic bacterium, designated HS1(T), was isolated from offshore hot spring sediment in Xiamen, China. Cells were Gram-negative, catalase-positive, oxidase-negative, slender and flexible rods without flagella. The strain could grow at 35-55 °C (optimum at 50 °C) and in 1-8 % NaCl (w/v; optimum 2-4 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HS1(T) was affiliated with the family Marinilabiliaceae and shared a distant relationship with the previously described genera. The isolate was most closely related to Anaerophaga thermohalophila Fru22(T) with 16S rRNA gene sequence similarity of 92.4 %, followed by the other members of the family Marinilabiliaceae with 88.7-91.1 % similarity. The dominant cellular fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). The predominant quinone was MK-7. The major polar lipids were phosphatidylethanolamine (PE) and an unknown polar lipid. The genomic DNA G+C content was 38.7 mol%. Besides the phylogenetically distant relationship, strain HS1(T) was obviously distinguished from the most closely related genera in several phenotypic properties including colony colour and pigment production, optimal temperature, optimal NaCl, relation to O(2), bicarbonate/carbonate requirement, catalase activity, nitrate reduction, fermentation products and cellular fatty acid profile. Based on the phenotypic and phylogenetic data, strain HS1(T) represents a novel species of a new genus, for which the name Thermophagus xiamenensis gen. nov., sp. nov. is proposed. The type strain of the type species is HS1(T) (= DSM 19012(T) = CGMCCC 1.5071(T)).

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.

Purpose: Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites., Materials and Methods: Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present., Results: Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes and Spirochaetes. Dialister invisus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis, Prevotella denticola, Tannerella forsythia, and Treponema denticola, which have been associated with periodontal disease, were found in significantly greater abundance in pericoronal compared with incisor sites. Dialister invisus and F nucleatum were found in greater abundance in sites exhibiting clinical symptoms. The archaeal species, Methanobrevibacter oralis, which has been associated with severe periodontitis, was found in 3 symptomatic patients., Conclusions: These findings have provided new insights into the complex microbiota of pericoronitis. Several bacterial and archaeal species implicated in periodontal disease were recovered in greater incidence and abundance from the plaque of partially erupted third molars compared with incisors, supporting the hypothesis that the pericoronal region may provide a favored niche for periodontal pathogens in otherwise healthy mouths., (Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg(2+) ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.

A novel mesophilic member of the Thermotogales, strain MesG1.Ag.4.2, was isolated from sediments from Baltimore Harbor, MD, USA. The strain grew optimally at 37 °C with a doubling time of 16.5 h on xylose. Carbohydrates and proteinaceous compounds supported growth and pentoses were preferred over hexoses. The strain was strictly anaerobic and growth was slightly stimulated by thiosulfate, sulfite, and elemental sulfur. The G + C content of its genomic DNA was 45.3 mol%. Strain MesG1.Ag.4.2 and Kosmotoga olearia lipids were analyzed. Strain MesG1.Ag.4.2 contained no long-chain dicarboxylic acids and its major phospholipid was lyso-phosphatidylserine. Long-chain dicarboxylic acids were found in K. olearia and its major phospholipid was cardiolipin, a lipid not yet reported in Thermotogales species. Phylogenetic analyses of its two 16S rRNA genes placed strain MesG1.Ag.4.2 within the bacterial order Thermotogales. Based on the phylogenetic analyses and its low optimal growth temperature, it is proposed that the strain represents a novel species of a new genus within the family Thermotogaceae, order Thermotogales. The name Mesotoga prima gen. nov., sp. nov. is proposed. The type strain of M. prima is MesG1.Ag.4.2 (= DSM 24739 = ATCC BAA-2239).

Endo-β-1,4-glucanase from thermophilic Fervidobacterium nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase family 5, is highly thermostable and exhibits the highest activity on carboxymethylcellulose among the reported homologues. To understand the structural basis for the thermostability and catalytic mechanism, we report here the crystal structures of FnCel5A and the complex with glucose at atomic resolution. FnCel5A exhibited a (β/α)(8)-barrel structure typical of clan GH-A of the glycoside hydrolase families with a large and deep catalytic pocket located in the C-terminal end of the β-strands that may permit substrate access. A comparison of the structure of FnCel5A with related structures from thermopile Clostridium thermocellum, mesophile Clostridium cellulolyticum, and psychrophile Pseudoalteromonas haloplanktis showed significant differences in intramolecular interactions (salt bridges and hydrogen bonds) that may account for the difference in their thermostabilities. The substrate complex structure in combination with a mutagenesis analysis of the catalytic residues implicates a distinctive catalytic module Glu(167)-His(226)-Glu(283), which suggests that the histidine may function as an intermediate for the electron transfer network between the typical Glu-Glu catalytic module. Further investigation suggested that the aromatic residues Trp(61), Trp(204), Phe(231), and Trp(240) as well as polar residues Asn(51), His(127), Tyr(228), and His(235) in the active site not only participated in substrate binding but also provided a unique microenvironment suitable for catalysis. These results provide substantial insight into the unique characteristics of FnCel5A for catalysis and adaptation to extreme temperature.

Four hyperthermophilic members of the bacterial genus Thermotoga (T. maritima, T. neapolitana, T. petrophila, and Thermotoga sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptomic analysis based on a multispecies cDNA microarray. Growth on a mixture of six pentoses and hexoses showed no significant utilization of galactose or mannose by any of the four species. T. maritima and T. neapolitana exhibited similar monosaccharide utilization profiles, with a strong preference for glucose and xylose over fructose and arabinose. Thermotoga sp. strain RQ2 also used glucose and xylose, but was the only species to utilize fructose to any extent, consistent with a phosphotransferase system (PTS) specific for this sugar encoded in its genome. T. petrophila used glucose to a significantly lesser extent than the other species. In fact, the XylR regulon was triggered by growth on glucose for T. petrophila, which was attributed to the absence of a glucose transporter (XylE2F2K2), otherwise present in the other Thermotoga species. This suggested that T. petrophila acquires glucose through the XylE1F1K1 transporter, which primarily serves to transport xylose in the other three Thermotoga species. The results here show that subtle differences exist among the hyperthermophilic Thermotogales with respect to carbohydrate utilization, which supports their designation as separate species.

Background: Thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp., Results: Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure., Conclusions: Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

Hydrogenotrophic microbiota have a significant impact on colonic health; however, little is known about their diversity and ecology in situ. Here, molecular-based methods and multivariate analyses were used to examine the abundance and diversity of mucosa-associated hydrogenotrophic microbes in 90 biopsies collected from right colon, left colon and rectum of 25 healthy subjects. Functional genes of all three hydrogenotrophic groups were detected in at least one colonic region of all subjects. Methanogenic archaea (MA) constituted approximately one half of the hydrogenotrophic microbiota in each colonic region. Sulfate-reducing bacteria (SRB) were more abundant than acetogens in right colon, while acetogens were more abundant than SRB in left colon and rectum. MA genotypes exhibited low diversity, whereas SRB genotypes were diverse and generally similar across the three regions within subject but significantly variable among subjects. Multivariate cluster analysis defined subject-specific patterns for the diversity of SRB genotypes; however, neither subject- nor region-specific clusters were observed for the abundance of hydrogenotrophic functional genes. Sequence analyses of functional gene clones revealed that mucosa-associated SRB were phylogenetically related to Desulfovibrio piger, Desulfovibrio desulfuricans and Bilophila wadsworthia; whereas MA were related to Methanobrevibacter spp., Mb. smithii and the order Methanomicrobiales. Together these data demonstrate for the first time that the human colonic mucosa is persistently colonized by all three groups of hydrogenotrophic microbes, which exhibit segmental and interindividual variation in abundance and diversity.

Quantitative characterization of the mode and rate of phenotypic evolution is rarely applied to prokaryotes. Here, we present an analysis of temperature optimum (T (opt)) evolution in the thermophilic family Thermotogaceae, which has a large number of cultured representatives. We use log-rate-interval analysis to show that T (opt) evolution in Thermotogaceae is consistent with a Brownian motion (BM) evolutionary model. The properties of the BM model are used to a establish confidence intervals on the unknown phenotypic trait value of an uncultured organism, given its distance to a close relative with known trait value. Cross-validation by bootstrapping indicates that the predictions are robust.

An anaerobic rod-shaped thermophile was isolated from a hydrothermal vent at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean, and was named strain MN14(T). The rods were gram-negative-staining, non-motile without flagella, 2-4 µm long and 0.5 µm wide, and divided by binary fission in the mid-exponential phase. The cells were surrounded by a sheath-like structure (toga) and occurred singly or in chains. Spheroids containing multiple cells were observed not only in the stationary phase, as previously observed for species of the order Thermotogales, but also from the early exponential phase. Transmission electron microscopy revealed that the peptidoglycan in rods partly disintegrated in the early growth phases and that the outer membrane of the spheroids was not completely lined with peptidoglycan. These findings suggested that the spheroids were formed from rods by the disintegration of peptidoglycan and subsequent inflation of the outer membrane. The spheroids eventually generated tiny cells in the periplasmic space, indicating a viviparous mode of proliferation in addition to binary fission. Strain MN14(T) grew at 40-75 °C, pH 5.0-8.2 and with 0.25-5.20 % (w/v) NaCl, with optimal growth occurring at 68 °C, pH 6.8 and with 2.5 % NaCl. The shortest doubling time was 24 min, assuming that the strain propagated only by binary fission. Elemental sulfur enhanced growth, but was not essential. Thiosulfate was not an electron acceptor for growth. The strain was a chemo-organotroph that grew on yeast extract as the sole growth substrate. Tryptone and starch supported its growth in the presence of yeast extract. The G+C content of the genomic DNA was 31.7 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Thermosipho. No significant DNA-DNA hybridization was observed between the genomic DNA of strain MN14(T) and phylogenetically related species of the genus Thermosipho. Based on this evidence, strain MN14(T) is proposed to represent a novel species, named Thermosipho globiformans sp. nov. The species epithet globiformans reflects the formation of multicellular and reproductive spheroids by the novel strain. The type strain of this species is MN14(T) ( = JCM 15059(T) = DSM 19918(T)).

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

A bioinformatic screening of the genome of the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 for esterhydrolyzing enzymes revealed a putative bacterial esterase (FNE) encoded by Fond&#95;1301 with typical GDSL family motifs. To confirm its putative esterase function, the FNE gene was cloned, functionally expressed in Escherichia coli, and purified to homogeneity. Recombinant FNE exhibited the highest esterase activity of 14,000 U/mg with p-nitrophenyl acetate (pNPC(2)) as substrate. The catalytic efficiency (k(cat)/K(m)) toward p-nitrophenyl acetate (C(2)) was approximately 120-fold higher than toward p-nitrophenyl butyrate (C(4)). No significant esterase activity was observed for the substrates with a chain length > or =C(8). The monomeric enzyme has a molecular mass of 27.5 kDa and exhibits optimal activity around 75 degrees C, at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70 degrees C, but less stable compared with some other hyperthermophilic esterases. A structural model was constructed using acetylesterase from Aspergillus aculeatus as a template. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine, which was verified by site-directed mutagenesis. Sequence analysis showed that FNE was only distantly related to other esterases. A comparison of the conserved motifs shared with GDSL proteins revealed that FNE could be grouped into GDSL family and was further classified as SGNH hydrolase.

The present article gives an overview of recent taxonomic changes among the Gram-negative, anaerobic rods, briefly highlighting areas where the biology and ecology have a bearing on recent nomenclatorial changes. The focus is among the genera Bacteroides, Prevotella, Porphyromonas, Leptotrichia, Dysgonomonas, Fusobacterium and the Synergistes group and additionally demonstrates the value of conserved indels and group-specific proteins for identifying and circumscribing many of these taxa and the Bacteroidetes-Chlorobi species in general.

Background: Recent data have shown that periodontal disease may increase the risk of occurrence of coronary heart disease in which inflammation initiated by bacteria and their compounds might be a common causal factor. This case-control study aimed at studying the relationship between periodontal disease and coronary artery disease (CAD) based on clinical and periodontal microbiologic parameters., Methods: A total of 90 male subjects, 48 to 80 years of age, were included in this study. Forty-five men had CAD (CAD+), which was confirmed by coronary angiography. Forty-five age-matched controls showed no history or symptoms of CAD (CAD-). All subjects underwent a clinical periodontal examination including assessment of tooth loss, probing depth, clinical attachment level, and bleeding on probing. In the CAD+ group, this examination took place 1 day before coronary angiography. Subgingival microbial samples were taken and evaluated by means of real-time polymerase chain reaction (RT-PCR) for the total amount of bacteria and the following periodontopathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra (formerly Micromonas micros), Dialister pneumosintes, and Campylobacter rectus., Results: Compared to control subjects, CAD+ subjects had significantly deeper pockets (2.28 mm versus 2.96 mm; P <0.001) and greater attachment loss (2.85 mm versus 3.65 mm; P <0.001), and this difference remained statistically significant after adjusting for smoking. No significant differences were observed between cases and controls with regard to the number of teeth present. P. intermedia was the only periodontal pathogen that showed significantly higher mean counts in CAD+ subjects compared to CAD- subjects. Higher counts of total bacteria, P. micra, D. pneumosintes, and C. rectus were found in the CAD- group., Conclusion: The results suggest that a relationship between periodontal disease and coronary heart disease exists, although P. intermedia was the only periodontopathogen related to CAD.

A novel thermophilic and sulfur-reducing heterotrophic bacterium, strain TFS10-5(T), was isolated from a deep-sea hydrothermal field in Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain TFS10-5(T) were motile rods, 1.5-5 microm in length and 0.5-0.8 microm in width. Strain TFS10-5(T) was an obligately anaerobic heterotroph and sulfur-reduction stimulated growth. Growth was observed between 30 and 70 degrees C (optimum at 55-60 degrees C), pH 5.0-7.4 (optimum at pH 5.5-5.8), 1.0-5.5 NaCl % (optimum at 3.0-3.5 %). The fatty acid content was C(16 : 0) (71.0 %), C(16 : 1) (6.0 %), C(18 : 0) (21.4 %) and C(18 : 1) (1.6 %). The G+C content of the genomic DNA was 28 mol%. 16S rRNA gene sequence analysis indicated that strain TFS10-5(T) belongs to the genus Marinitoga. Based on the physiological and phylogenetic features of the new isolate, strain TFS10-5(T) represents a novel species in the genus Marinitoga for which the name Marinitoga okinawensis sp. nov. is proposed. The type strain is TFS10-5(T) (=JCM 13303(T)=DSM 17373(T)).

A novel thermophilic, moderately halophilic, rod-shaped bacterium, strain MET-B(T), with a sheath-like outer structure (toga) was isolated from an offshore oil-producing well in Congo, West Africa. Strain MET-B(T) was a Gram-negative bacterium with the ability to reduce elemental sulfur, but not sulfate, thiosulfate or sulfite into sulfide. The optimum growth conditions were 60 degrees C, pH 6.7-7.2 and 4-6 % NaCl. The DNA G+C content was 34.6 mol%. Strain MET-B(T) was phylogenetically related to members of the genus Petrotoga; Petrotoga miotherma, Petrotoga olearia and Petrotoga mexicana were the closest relatives, with type strains exhibiting more than 99 % identity in an analysis of small-subunit rRNA gene sequences. The values for DNA-DNA relatedness between the type strains of these three species and strain MET-B(T) were less than 42 %. As MET-B(T) was found to be genetically and physiologically different from other species of the genus Petrotoga, this strain is proposed as representing a novel species, for which the name Petrotoga halophila sp. nov. is proposed. The type strain is MET-B(T) (=DSM 16923(T)=CCUG 50214(T)).