Genomic and metabolic analysis of Komagataeibacter xylinus DSM 2325 producing bacterial cellulose nanofiber.

Bacterial cellulose nanofiber (CNF) is a polymer with a wide range of potential industrial applications. Several Komagataeibacter species, including Komagataeibacter xylinus as a model organism, produce CNF. However, the industrial application of CNF has been hampered by inefficient CNF production, necessitating metabolic engineering for the enhanced CNF production. Here, we present complete genome sequence and a genome-scale metabolic model KxyMBEL1810 of K. xylinus DSM 2325 for metabolic engineering applications. Genome analysis of this bacterium revealed that a set of genes associated with CNF biosynthesis and regulation were present in this bacterium, which were also conserved in another six representative Komagataeibacter species having complete genome information. To better understand the metabolic characteristics of K. xylinus DSM 2325, KxyMBEL1810 was reconstructed using genome annotation data, relevant computational resources and experimental growth data generated in this study. Random sampling and correlation analysis of the KxyMBEL1810 predicted pgi and gnd genes as novel overexpression targets for the enhanced CNF production. Among engineered K. xylinus strains individually overexpressing heterologous pgi and gnd genes, either from Escherichia coli or Corynebacterium glutamicum, batch fermentation of a strain overexpressing the E. coli pgi gene produced 3.15 g/L of CNF in a complex medium containing glucose, which was the best CNF concentration achieved in this study, and 115.8% higher than that (1.46 g/L) obtained from the control strain. Genome sequence data and KxyMBEL1810 generated in this study should be useful resources for metabolic engineering of K. xylinus for the enhanced CNF production.

Bioactive bacterial cellulose sulfate electrospun nanofibers for tissue engineering applications.

Cellulosic materials have been of tremendous importance to mankind since its discovery due to its superior properties and its abundance in nature. Recently, an increase in demand for alternate green materials has rekindled the interest for cellulosic materials. Here, bacterial cellulose has been functionalized with sulfate groups through acetosulfation to gain solubility in aqueous media, which provides access to several applications. The cell viability, antioxidant, and hemocompatibility assays have verified the biocompatible and antioxidant characteristics of bacterial cellulose sulfate (BCS) in both in vitro and ex vivo conditions. Further, novel BCS/polyvinyl alcohol nanofibers were fabricated by simple electrospinning route to engineer ultrafine nanoscale fibers. The biological evaluation of BCS/polyvinyl alcohol nanofiber scaffolds was done using L929 mouse fibroblast cells, which confirmed that these nanofibers are excellent matrices for cell adhesion and proliferation.

Crystallization and preliminary X-ray diffraction analysis of the secreted protein Athe_0614 from Caldicellulosiruptor bescii.

The Athe_0614 protein is a component of the extracellular proteins secreted by the anaerobic, extremely thermophilic and cellulolytic bacterium Caldicellulosiruptor bescii. The recombinant protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized using polyethylene glycol 2000 monomethyl ether as a precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 48.4, b = 42.2, c = 97.8 Å, ß = 96.1°, and diffracted to 2.7 Å resolution using synchrotron radiation.

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus.

The optimum conditions for the production of L-arabinose from debranched arabinan were determined to be pH 6.5, 75°C, 20 g l(-1) debranched arabinan, 42 Um l(-1) endo-1,5-α-L-arabinanase, and 14 U ml(-1) α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75°C, 20 g l(-1) sugar beet arabinan, 3 U ml(-1) endo-1,5-α-L-arabinanase, and 24 U ml(-1) α-L-arabinofuranosidase. Under the optimum conditions, 16 g l(-1)l-arabinose was obtained from 20 g l(-1) debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l(-1)h(-1). This is the first reported trial for the production of L-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.

Carboxydothermus siderophilus sp. nov., a thermophilic, hydrogenogenic, carboxydotrophic, dissimilatory Fe(III)-reducing bacterium from a Kamchatka hot spring.

A novel anaerobic, thermophilic, Fe(III)-reducing, CO-utilizing bacterium, strain 1315(T), was isolated from a hot spring of Geyser Valley on the Kamchatka Peninsula. Cells of the new isolate were Gram-positive, short rods. Growth was observed at 52-70 degrees C, with an optimum at 65 degrees C, and at pH 5.5-8.5, with an optimum at pH 6.5-7.2. In the presence of Fe(III) or 9,10-anthraquinone 2,6-disulfonate (AQDS), the bacterium was capable of growth with CO and yeast extract (0.2 g l(-1)); during growth under these conditions, strain 1315(T) produced H(2) and CO(2) and Fe(II) or AQDSH(2), respectively. Strain 1315(T) also grew by oxidation of yeast extract, glucose, xylose or lactate under a N(2) atmosphere, reducing Fe(III) or AQDS. Yeast extract (0.2 g l(-1)) was required for growth. Isolate 1315(T) grew exclusively with Fe(III) or AQDS as an electron acceptor. The generation time under optimal conditions with CO as growth substrate was 9.3 h. The G+C content of the DNA was 41.5+/-0.5 mol%. 16S rRNA gene sequence analysis placed the organism in the genus Carboxydothermus (97.8 % similarity with the closest relative). On the basis of physiological features and phylogenetic analysis, it is proposed that strain 1315(T) should be assigned to a novel species, Carboxydothermus siderophilus sp. nov., with the type strain 1315(T) (=VKPM 9905B(T) =VKM B-2474(T) =DSM 21278(T)).

Ammonifex thiophilus sp. nov., a hyperthermophilic anaerobic bacterium from a Kamchatka hot spring.

A novel anaerobic, extremely thermophilic, facultatively chemolithoautotrophic bacterium designated strain SR(T) was isolated from a terrestrial hot spring in Kamchatka (Russia). The cells of the novel strain were spore-forming rods with a Gram-positive type of cell wall. The novel isolate grew at 60-82 degrees C (optimum 75 degrees C) and pH 6.0-7.5 (optimum 6.8). Molecular hydrogen and formate were used as electron donors. Thiosulfate, sulfate or elemental sulfur served as electron acceptors yielding hydrogen sulfide. No growth was observed on either substrate in the presence of nitrate as the electron acceptor. The G+C content of the DNA was 56.2 mol%. Phylogenetic analyses of the 16S rRNA gene showed that strain SR(T) was most closely related to Ammonifex degensii (96.4 % gene sequence similarity). However, the novel isolate possessed phenotypic characteristics that differed significantly from those of A. degensii, the only other recognized species of the genus Ammonifex. It is concluded that strain SR(T) (=DSM 19636(T)=VKM B-2461(T)) represents the type strain of a novel species of the genus Ammonifex, for which the name Ammonifex thiophilus sp. nov. is proposed. An emendation of the genus Ammonifex is proposed based on the phenotypic characteristics of the novel species.

Xenon in and at the end of the tunnel of bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase.

A fascinating feature of some bifunctional enzymes is the presence of an internal channel or tunnel to connect the multiple active sites. A channel can allow for a reaction intermediate generated at one active site to be used as a substrate at a second active site, without the need for the intermediate to leave the safety of the protein matrix. One such bifunctional enzyme is carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica (mtCODH/ACS). A key player in the global carbon cycle, CODH/ACS uses a Ni-Fe-S center called the C-cluster to reduce carbon dioxide to carbon monoxide and uses a second Ni-Fe-S center, called the A-cluster, to assemble acetyl-CoA from a methyl group, coenzyme A, and C-cluster-generated CO. mtCODH/ACS has been proposed to contain one of the longest enzyme channels (138 A long) to allow for intermolecular CO transport. Here, we report a 2.5 A resolution structure of xenon-pressurized mtCODH/ACS and examine the nature of gaseous cavities within this enzyme. We find that the cavity calculation program CAVENV accurately predicts the channels connecting the C- and A-clusters, with 17 of 19 xenon binding sites within the predicted regions. Using this X-ray data, we analyze the amino acid composition surrounding the 19 Xe sites and consider how the protein fold is utilized to carve out such an impressive interior passageway. Finally, structural comparisons of Xe-pressurized mtCODH/ACS with related enzyme structures allow us to study channel design principles, as well as consider the conformational flexibility of an enzyme that contains a cavity through its center.

Tepidimicrobium ferriphilum gen. nov., sp. nov., a novel moderately thermophilic, Fe(III)-reducing bacterium of the order Clostridiales.

A moderately thermophilic, anaerobic bacterium (strain SB91T) was isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia. Cells of strain SB91T were straight to slightly curved rods, 0.5-0.6 microm in diameter and 3.0-7.0 mum in length. Formation of endospores was not observed. The temperature range for growth was 26-62 degrees C, with an optimum at 50 degrees C. The pH range for growth was 5.5-9.5, with an optimum at pH 7.5-8.0. The substrates utilized by strain SB91T in the presence of 9,10-anthraquinone 2,6-disulfonate included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, alanine plus glycine, alanine plus proline, L-valine and n-propanol. Carbohydrates were not utilized. Strain SB91T reduced amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA or Fe(III) nitrilotriacetate with peptone, L-valine or n-propanol as an electron donor. Strain SB91T reduced 9,10-anthraquinone 2,6-disulfonate, thiosulfate, elemental sulfur, fumarate and selenite. Strain SB91T survived after exposure to gamma-radiation at a dose of 5.4 kGy. The G+C content of the DNA of strain SB91T was 33 mol%. Analysis of the 16S rRNA gene sequence revealed that the isolated organism belonged to cluster XII of the clostridia. On the basis of its physiological properties and the results of phylogenetic analyses, it is proposed that strain SB91T represents the sole species of a novel genus, Tepidimicrobium; the name Tepidimicrobium ferriphilum gen. nov., sp. nov. is proposed, with strain SB91T (=DSM 16624T=VKM B-2348T) as the type strain.

Leucobacter luti sp. nov., and Leucobacter alluvii sp. nov., two new species of the genus Leucobacter isolated under chromium stress.

Two strains designated RF6(T) and RB10(T) were isolated, from activated sludge and from river sediments, respectively, both systems receiving chromium contaminated water. Phylogenetic analysis showed that strain RF6(T)and strain RB10(T) represented two new species of the genus Leucobacter. Strain RB10(T) can be distinguished from RF6(T) by its ability to grow at 37 degrees C, by showing a different optimum pH, by cell wall amino acids different relative amount and by having the fatty acid strait C16:0 as the third most abundant fatty acid. On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain RF6(T) represents a new species of the genus Leucobacter for which we propose the name Leucobacter luti (CIP 108818(T)=LMG 23118) and that strain RB10(T) represents an additional new species of the same genus for which we propose the name Leucobacter alluvii (CIP 108819(T)=LMG 23117).

X-ray crystal structures of Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric oxide reductase.

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent-bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fe1-Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 A with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N(2)O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction.

Characterization of Carnobacterium divergens strain 6251 isolated from intestine of Arctic charr (Salvelinus alpinus L.).

An atypical strain of Carnobacterium divergens, strain 6251, was isolated from the small intestine of Arctic charr (Salvelinus alpinus L.), fed high dietary carbohydrate. This strain showed marked growth inhibitory effects in vitro against the fish pathogens Aeromonas salmonicida subsp. salmonicida (furunculosis), Vibrio anguillarum (vibriosis) and Vibrio viscocus (winter ulcer). The strain is a non-motile Gram-positive psychrotrophic rod that lacks both catalase and oxidase, grows at pH 9.1 (CTAS agar), but not on acetate containing media (pH < or = 5.4), on TCBS or at < or =6% sodium chloride content. Strain 6251 is facultatively anaerobic and utilises tryptone as a sole source of nutrient. Further characterisation showed the most abundant cellular fatty acid of strain 6251 to be oleic acid (18:1) (n-9) (36.0%). Sequencing of a 16S rDNA region of 578 nucleotides and AFLP microbial fingerprinting suggested that strain 6251 is not closely related to any carnobacteria known, however, DNA-DNA similarity determinations showed high similarity (96.2%) with the type strain of Carnobacterium divergens. The unique phenotypic attributes of this strain represent new information on the biodiversity and ecology of carnobacteria and especially of the species C. divergens.

Transcriptional analysis of the xylose ABC transport operons in the thermophilic anaerobe Thermoanaerobacter ethanolicus.

Thermoanaerobacter ethanolicus is a Gram-positive thermophile that converts xylose to ethanol. A portion of the T. ethanolicus xylose transport permease gene ( xylH) was cloned, and the deduced protein exhibited greater than 60% similarity to homologs in enterobacteria. Xylose-binding protein ( xylF) and ( xylH) transcripts were quantitated and compared from cells grown in batch or continuous cultures grown on xylose, glucose, or a mixture of both sugars. In contrast to the strong repression of xyl operons by glucose in other bacteria, both xylF and xylH expression were detected in the presence of this hexose sugar. Expression of xylF and xylH generally increased with dilution rate (3- and 1.5-fold, respectively) and seemed to be growth rate rather than substrate dependent. Overall, these unusual sugar utilization patterns in batch and continuous culture seem to result from a basal expression level of xyl genes in the absence of xylose. T. ethanolicus is unique in possessing a triumvirate of xylose transport and catabolism operons and, given its extensive hemicellulolytic capabilities, may have evolved to constitutively express xyl genes.

Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797.

Contamination of feed with trichothecenes, a group of Fusarium mycotoxins, leads to losses in performance due to their immunosupressive effects and the negative effect on the gastrointestinal system in animal production. A possible way of detoxification is microbial degradation, which was the focus of this study. A bacterial strain--BBSH 797--which can degrade some mycotoxins of the trichothecene group, has already been isolated. It transforms deoxynivalenol (DON) into its metabolite DOM-1, the non-toxic deepoxide of DON. Analogous to the microbial degradation of DON, the transformation of six different type A trichothecenes was observed. The metabolites appearing were characterized by GC-MS after derivatization with TRI-SIL TBT. Two metabolites were additionally, identified by liquid chromatography-mass spectrometry with particle beam interface (LC-PB-MS) with electron impact (EI)-ionization mode. The major finding was that scirpentriol was completely transformed into its non-toxic metabolite deepoxy scirpentriol, while the mycotoxin T-2 triol underwent a more complicated metabolism. According to the study, T-2-triol was degraded into its non-toxic deepoxy form and into T-2 tetraol, which was then further metabolized to deepoxy T-2 tetraol. GC-MS after derivatization with TRI-SIL TBT was suitable for the structural characterization of trichothecenes and their degradation products. Besides the mass spectra of already known degradation products, spectra of new metabolites could be recorded by LC-PB-MS.

Time-dependent XAS studies of trapped enzyme-substrate complexes of alcohol dehydrogenase from Thermoanaerobacter brockii.

The understanding of structure-function relationships in proteins has been significantly advanced with the advent of the biotechnological revolution. A goal yet to be realized for many metalloenzyme systems is to characterize the dynamic changes in structure that bridge the static endpoints provided by crystallography. We present here a series of edge and EXAFS spectra of the metalloenzyme alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) complexed with its substrate. The enzyme-substrate complexes were trapped by fast freezing at various times, following their enzyme activity. Our edge and EXAFS analyses both reveal the time-dependent changes in the structure of the active site of TbADH.

Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200.

The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67-69% identity with alcohol dehydrogenases from two archaeal species and 29-37% identity with bacterial type III alcohol dehydrogenases. This represents the first reported cloning of an alcohol dehydrogenase from a bacterial species that is both thermostable and active against primary long-chain alcohols.

High resistance to oxygen radicals and heat is caused by a galactoglycerolipid in Microbacterium sp. M874.

Microbacterium sp. M874 produced a glyceroglycolipid, di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol, at about the 50 microM level. Though the strain was highly resistant to tertiary-butyl hydroperoxide (tBHP) in a glycolipid-productive medium, the resistance was reduced in a nonproductive medium. Exogenous addition of the glycolipid to the nonproductive culture restored the resistance. This addition also increased the resistance to heat, ethanol, and 4-chloro-1-naphthol, in which oxygen radicals might participate. The parallel relationship found in strain M874 mutants between glycolipid productivity and resistance to tBHP or heat suggested that the resistance was mainly caused by the glycolipid. On addition of the glycolipid to a glycolipid-nonproductive culture, it was immediately incorporated into the cells and functioned as an anti-oxygen radical reagent. Thereafter, its intracellular level remained largely unchanged for at least 5 h, even in the presence of tBHP, and its activity was maintained. The glycolipid at 142 microM was sufficient to prevent the cytotoxicity induced by 88.9 mM tBHP. The glycolipid production was not induced by pretreatment with a low level of tBHP or a sublethal heat shock. In brief, the glycolipid might play an essential role in the prevention of damage by oxygen radicals in the glycolipid-producing bacterium.

Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile thermoanaerobacter ethanolicus.

The gene for the bifunctional xylosidase-arabinosidase (xarB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequenced, and expressed in Escherichia coli (Genebank Accession No. AF135015). Analysis of the recombinant enzyme revealed activity against multiple substrates with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (pNPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside (pNPAP), respectively. Thus, we classify this enzyme as a bifunctional xylosidase-arabinosidase. Even though both sequences are 96% identical on the amino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T. brockii ATCC 33075 and a deletion in a downstream open reading frame in T. ethanolicus seem to have occurred through evolutionary divergence of these two species. This represents an interesting phenomenon of molecular evolution of bacterial species, as PCR analysis of the region around the deletion indicates that the deletion is not present in T. brockii ssp. finnii and T. brockii ssp. brockii type strain HTD4.

High-affinity maltose binding and transport by the thermophilic anaerobe Thermoanaerobacter ethanolicus 39E.

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [(14)C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and alpha-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and alpha-trehalose.

Organization and sequence of histidine biosynthesis genes hisH, -A, -F, and -IE in Thermoanaerobacter ethanolicus.

Nucleotide sequence analysis of a 3.5-kb chromosomal fragment from the low G + C Gram-positive bacterium Thermoanaerobacter ethanolicus revealed a cluster of five contiguous open reading frames (ORFs) designated hisH, hisA, hisF, hisIE, and ORF5. The first four ORFs showed homology to genes of the histidine biosynthesis pathway, and ORF5 encoded a product with no significant similarities to polypeptides presently known. The hisH ORF was partial (truncated by cloning) and ORF5 was adjacent to xylF, which codes for a xylose-binding periplasmic protein. The five genes encoded putative proteins of >104, 237, 254, 216, and 169 amino acids, respectively. Amino acid sequence comparison of the four his gene products indicated closely related homologs in prokaryotes, varying from low G + C Gram-positive bacteria to archaea. This is the first report of his anabolic genes in a thermophilic anaerobic bacterium.

Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium.

A thermophilic, anaerobic, spore-forming, dissimilatory Fe(III)-reducing bacterium, designated strain SR4T, was isolated from sediment of newly formed hydrothermal vents in the area of the eruption of Karymsky volcano on the Kamchatka peninsula. Cells of strain SR4T were straight-to-curved, peritrichous rods, 0.4-0.6 micron in diameter and 3.5-9.0 microns in length, and exhibited a slight tumbling motility. Strain SR4T formed round, refractile, heat-resistant endospores in terminally swollen sporangia. The temperature range for growth was 39-78 degrees C, with an optimum at 69-71 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.3-6.5. Strain SR4T grew anaerobically with peptone as carbon source. Amorphous iron(III) oxide present in the medium stimulated the growth of strain SR4T; cell numbers increased with the concomitant accumulation of Fe(II). In the presence of Fe(III), strain SR4T grew on H2/CO2 and utilized molecular hydrogen. Strain SR4T reduced 9,10-anthraquinone-2,6-disulfonic acid, sulfite, thiosulfate, elemental sulfur and MnO2. Strain SR4T did not reduce nitrate or sulfate and was not capable of growth with O2. The fermentation products from glucose were ethanol, lactate, H2 and CO2. The G + C content of DNA was 32 mol%. 16S rDNA sequence analysis placed the organism in the genus Thermoanaerobacter. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain SR4T (= DSM 12299T) should be assigned to a new species, Thermoanaerobacter siderophilus sp. nov.