Candidatus Mcinerneyibacterium aminivorans gen. nov., sp. nov., the first representative of the candidate phylum Mcinerneyibacteriota phyl. nov. recovered from a high temperature, high salinity tertiary oil reservoir in north central Oklahoma, USA.

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 ARYD3T, 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.

Enhanced production, overexpression and characterization of a hyperthermophilic multimodular GH family 2 ß­glucuronidase (TpGUS) cloned from Thermotoga petrophila RKU-1T in a mesophilic host.

A multimodular hyperthermophilic ß­glucuronidase (TpGUS) from Thermotoga petrophila RKU-1T, belongs to glycoside hydrolase family 2 (GH2), was cloned and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Expression and production of extracellular TpGUS was enhanced through various specific cultivation and induction strategies. Extracellular TpGUS activity was improved by 3.44 and 7 fold in 4 × ZB medium induced with 0.5 mM IPTG and 100 mM lactose, respectively. The enzyme was purified to homogeneity with a single band of 65.6 kDa on SDS-PAGE, using two subsequent steps of anion exchange and hydrophobic interaction chromatography after heat precipitation (70 °C, 1 h). Optimal activity of TpGUS was observed at 95 °C and pH 6.0; and it displayed prodigious thermal stability over a temperature range of 50-85 °C for 12 h at pH 6.0-7.5. Km, Vmax, VmaxKm-1, kcat, and kcatKm-1 were calculated to be 0.7 mM, 227 mmol mg-1 min-1, 324.3 min-1, 164,492.7 s-1 and 234,989.6 mM-1 s-1, respectively using pNPGU as a substrate. Recombinant TpGUS exhibited favorable properties which make this a promising candidate for various biotechnological and pharmacological applications.

A metabolic and genomic assessment of sugar fermentation profiles of the thermophilic Thermotogales, Fervidobacterium pennivorans.

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, H2, and CO2). 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 WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module.

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologues. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor.

Mesotoga prima strain PhosAc3 is a mesophilic representative of the phylum Thermotogae comprising only fermentative bacteria so far. We show that while unable to ferment glucose, this bacterium is able to couple its oxidation to reduction of elemental sulfur. We demonstrate furthermore that M. prima strain PhosAc3 as well as M. prima strain MesG1 and Mesotoga infera are able to grow in syntrophic association with sulfate-reducing bacteria (SRB) acting as hydrogen scavengers through interspecies hydrogen transfer. Hydrogen production was higher in M. prima strain PhosAc3 cells co-cultured with SRB than in cells cultured alone in the presence of elemental sulfur. We propose that the efficient sugar-oxidizing metabolism by M. prima strain PhosAc3 in syntrophic association with a hydrogenotrophic sulfate-reducing bacterium can be extrapolated to all members of the Mesotoga genus. Genome comparison of Thermotogae members suggests that the metabolic difference between Mesotoga and Thermotoga species (sugar oxidation versus fermentation) is mainly due to the absence of the bifurcating [FeFe]-hydrogenase in the former. Such an obligate oxidative process for using sugars, unusual within prokaryotes, is the first reported within the Thermotogae. It is hypothesized to be of primary ecological importance for growth of Mesotoga spp. in the environments that they inhabit.

Genomic insights into temperature-dependent transcriptional responses of Kosmotoga olearia, a deep-biosphere bacterium that can grow from 20 to 79 °C.

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.

Photobiosynthesis of stable and functional silver/silver chloride nanoparticles with hydrolytic activity using hyperthermophilic ß-glucosidases with industrial potential.

The ß-glucosidases are important enzymes employed in a large number of processes and industrial applications, including biofuel production from biomass. Therefore, in this study, we reported for the first time the photobiosynthesis of stable and functional silver/silver chloride nanoparticles (Ag/AgCl-NPs) using two hyperthermostable bacterial ß-glucosidases with industrial potential. The syntheses were straightforward and rapid processes carried out by mixing ß-glucosidase and silver nitrate (in buffer 10mM Tris-HCl, pH 8) under irradiation with light (over a wavelength range of 450-600nm), therefore, compatible with the green chemistry procedure. Synthesized Ag/AgCl-NPs were characterized using a series of physical techniques. Absorption spectroscopy showed a strong absorption band centered at 460nm due to surface plasmon resonance of the Ag-NPs. X-ray diffraction analysis revealed that the Ag/AgCl-NPs were purely crystalline in nature. Under electron microscopy, Ag/AgCl-NPs of variable diameter ranging from 10 to 100nm can be visualized. Furthermore, electron microscopy, zeta potential and Fourier transform infrared spectroscopy results confirmed the presence of ß-glucosidases coating and stabilizing the Ag/AgCl-NPs. Finally, the results showed that the enzymatic activities were maintained in the ß-glucosidases assisted Ag/AgCl-NPs. The information described here should provide a useful basis for future studies of ß-glucosidases assisted Ag/AgCl-NPs, including biotechnological applications.

Expression and Characterization of Hyperthermostable Exo-polygalacturonase TtGH28 from Thermotoga thermophilus.

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.

Natural transformation of Thermotoga sp. strain RQ7.

BACKGROUND: Thermotoga species are organisms of enormous interest from a biotechnological as well as evolutionary point of view. Genetic modifications of Thermotoga spp. are often desired in order to fully release their multifarious potentials. Effective transformation of recombinant DNA into these bacteria constitutes a critical step of such efforts. This study aims to establish natural competency in Thermotoga spp. and to provide a convenient method to transform these organisms. RESULTS: Foreign DNA was found to be relatively stable in the supernatant of a Thermotoga culture for up to 6 hours. Adding donor DNA to T. sp. strain RQ7 at its early exponential growth phase (OD600 0.18 ~ 0.20) resulted in direct acquisition of the DNA by the cells. Both T. neapolitana chromosomal DNA and Thermotoga-E. coli shuttle vectors effectively transformed T. sp. strain RQ7, rendering the cells resistance to kanamycin. The kan gene carried by the shuttle vector pDH10 was detected by PCR from the plasmid extract of the transformants, and the amplicons were verified by restriction digestions. A procedure for natural transformation of Thermotoga spp. was established and optimized. With the optimized method, T. sp. strain RQ7 sustained a transformation frequency in the order of 10⁻7 with both genomic and plasmid DNA. CONCLUSIONS: T. sp. strain RQ7 cells are naturally transformable during their early exponential phase. They acquire DNA from both closely and distantly related species. Both chromosomal DNA and plasmid DNA serve as suitable substrates for transformation. Our findings lend a convenient technical tool for the genetic engineering of Thermotoga spp.

A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis.

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.

Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales.

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).

Kinetic and thermodynamic study of cloned thermostable endo-1,4-ß-xylanase from Thermotoga petrophila in mesophilic host.

The 1,044 bp endo-1,4-ß-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 ß-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-ß-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.

Hyperthermophilic Thermotoga species differ with respect to specific carbohydrate transporters and glycoside hydrolases.

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.

Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough.

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)).

Methanol utilizing Desulfotomaculum species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor.

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l(-1), while on H2/CO2, no apparent inhibition occurred up to a concentration of 500 mg l(-1). When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l(-1). These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H2/CO2 to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.

Sedimenticola selenatireducens, gen. nov., sp. nov., an anaerobic selenate-respiring bacterium isolated from estuarine sediment.

The respiration of selenate, as a terminal electron acceptor has been known for over a decade, but the microorganisms involved in this respiration are largely unknown. Here we characterize a novel selenate-respiring bacterium, strain AK4OH1, isolated from an estuarine sediment enrichment culture. Strain AK4OH1 has the unique capability to oxidize aromatic acids, such as benzoate, 4-hydroxybenzoate and 3-hydroxybenzoate, coupled to selenate respiration. This novel respiratory coupling has not been described before. Reduction of selenate is followed by stoichiometric accumulation of selenite. The strain grows in agar shake tubes forming bright red colonies due to precipitation of elemental selenium. Strain AK4OH1 is a strictly anaerobic bacterium, which can also respire nitrate and nitrite via denitrification. Analysis of the 16S rRNA gene sequence shows that this strain clusters with another selenate-reducing bacterium and a (per) chlorate reducing bacterium, within the Gammaproteobacteria, along with symbionts of bivalves and tubeworms. Based on its unique physiological capabilities and its 16S rRNA gene sequence phylogeny, we classify this strain AK4OH1 as a new genus and species with the proposed name Sedimenticola selenatireducens.

H(2) production and carbon utilization by Thermotoga neapolitana under anaerobic and microaerobic growth conditions.

H(2) production by Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana was compared under microaerobic conditions. Contrary to these previously reported strains being strict anaerobes, all tested strains grew and produced H(2) in the presence of micromolar levels of O(2). T. neapolitana showed the highest H(2) production under these conditions. Microscopic counting techniques were used to determine growth curves and doubling times, which were subsequently correlated with optical density measurements. The Biolog anaerobic microtiter plate system was used to analyze the carbon source utilization spectrum of T. neapolitana and to select non-metabolized or poorly metabolized carbohydrates as physiological buffers. Itaconic acid was successfully used as a buffer to overcome pH-induced limitations of cell growth and to facilitate enhanced production of CO-free H(2).

Petrotoga mexicana sp. nov., a novel thermophilic, anaerobic and xylanolytic bacterium isolated from an oil-producing well in the Gulf of Mexico.

A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O. The DNA G+C content was 36.1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8%. However DNA-DNA relatedness values between these species and strain MET12T were lower than 70%. As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.

[Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: molekuliarnaia struktura lokusa.

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.