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Abstract: Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous (∼15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5α (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity. [Copyright &y& Elsevier]

Endospore formation is a specific property found within bacteria belonging to the Gram-type-positive low G+C mol% branch (Firmicutes) of a phylogenetic tree based on 16S rRNA genes. Within the Gram-type-positive bacteria, endospore-formers and species without observed spore formation are widely intermingled. In the present study, a previously reported experimental method (PCR and Southern hybridization assays) and analysis of genome sequences from 52 bacteria and archaea representing sporulating, non-spore-forming, and asporogenic species were used to distinguish non-spore-forming (void of the majority of sporulation-specific genes) from asporogenic (contain the majority of sporulation-specific genes) bacteria. Several sporulating species lacked sequences similar to those ofBacillus subtilissporulation genes. For some of the genes thought to be sporulation specific, sequences with weak similarity were identified in non-spore-forming bacteria outside of the Gram-type-positive phylogenetic branch and in archaea, rendering these genes unsuitable for the intended classification into sporulating, asporogenic, and non-spore-forming species. The obtained results raise questions regarding the evolution of sporulation among theFirmicutes. [ABSTRACT FROM AUTHOR]

The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature. [ABSTRACT FROM AUTHOR]

A correction to an article related to Thermosediminbacter that was published in a 2005 issue is presented.

The bacterium Natranaerobius thermophilus is an extremely halophilic alkalithermophile that can thrive under conditions of high salinity (3.3-3.9 M Na + ), alkaline pH (9.5), and elevated temperature (53°C). To understand the molecular mechanisms of salt adaptation in N. thermophilus , it is essential to investigate the protein, mRNA, and key metabolite levels on a molecular basis. Based on proteome profiling of N. thermophilus under 3.1, 3.7, and 4.3 M Na + conditions compared to 2.5 M Na + condition, we discovered that a hybrid strategy, combining the "compatible solute" and "salt-in" mechanisms, was utilized for osmotic adjustment dur ing the long-term salinity adaptation of N. thermophilus . The mRNA level of key proteins and the intracellular content of compatible solutes and K + support this conclusion. Specifically, N. thermophilus employs the glycine betaine ABC transporters (Opu and ProU families), Na + /solute symporters (SSS family), and glutamate and proline synthesis pathways to adapt to high salinity. The intracellular content of compatible solutes, including glycine betaine, glutamate, and proline, increases with rising salinity levels in N. thermophilus . Additionally, the upregulation of Na + / K + / H + transporters facilitates the maintenance of intracellular K + concentration, ensuring cellular ion homeostasis under varying salinities. Furthermore, N. thermophilus exhibits cytoplasmic acidification in response to high Na + concentrations. The median isoelectric points of the upregulated proteins decrease with increasing salinity. Amino acid metabolism, carbohydrate and energy metabolism, membrane transport, and bacterial chemotaxis activities contribute to the adaptability of N. thermophilus under high salt stress. This study provides new data that support further elucidating the complex adaptation mechanisms of N. thermophilus under multiple extremes.IMPORTANCEThis study represents the first report of simultaneous utilization of two salt adaptation mechanisms within the Clostridia class in response to long-term salinity stress., Competing Interests: The authors declare no conflict of interest.

The genus Natronolimnobius , currently including four species, is a member of the order Natrialbales , class Halobacteria, and consists of obligately alkaliphilic and extremely halophilic members found exclusively in highly alkaline hypersaline soda lakes. The species were classified into this genus mostly based on phylogenetic analysis of the 16S rRNA gene. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein markers clearly indicates a polyphyletic origin of the species included into this genus, thus warranting its reclassification into three separate genera. We therefore propose to transfer Nlb. innermongolicus (type strain N-1311) to a new genus Natronolimnohabitans as Nlh. innermongolicus comb. nov. and to transfer Nlb. aegyptiacus (type strain JW/NM-HA 15) and Nlb. sulfurireducens (type strain AArc1) to a new genus Natrarchaeobaculum as Nbl. aegyptiacum comb. nov. and Nbl. sulfurireducens comb. nov. The phylogenomic differentiation of these four species is also supported by the ANI/AAI distances and unique phenotypes. The most important physiological differences includes a previously unreported ability for cellulose and xylan utilization in Nlb. baerhuensis , thermophily in Nbl. aegyptiacus and anaerobic sulfur respiration in Nbl. sulfurireducens . We further present an emended description of Natronolimnobius baerhuensis .

A pullulanase, PulD7, was identified in the genome of the halophilic, alkalithermophilic isolate Alkalilimnicola sp. NM-DCM-1. PulD7 is 701 amino acids large with a carbohydrate binding module (CBM) 48 at the N-terminal. The full length PulD7 and N- and C-terminal truncated versions were cloned, heterologously expressed and functionally characterized. PulD7 displayed maximal activity at 55 °C, pH 9.5 and 2 M NaCl. PulD7 had good thermal stability, with a half-life of 693 min at 50 °C. PulD7 is an amylopullulanase, hydrolyzing both α‑1,4‑ and α‑1,6‑glycosidic bonds in soluble starch and pullulan, respectively. PulD7 was resistant to chemical reagents, including organic solvents (dimethyl sulfoxide, methanol, benzene, 20% v/v), reducing agents (β-mercaptoethanol, 5% v/v), surfactants (SDS and Tween 20, 5% v/v), the divalent chelator ethylene diamine tetra acetic acid (5 mM), and the chemical denaturant urea (8 M). PulD7 was not calcium-dependent. PulD7 was able to bind raw starch granules, reaching 52% binding in 3 h. The N-and C-terminal truncated forms of PulD7 had similar biochemical characteristics. PulD7ΔC had higher specific activity and halotolerance. The N-terminally truncated PulD7ΔN hydrolyzed amylose only, indicating that CBM48 is essential for binding branched substrates. PulD7 has unique characteristics giving it strong potential for application in biotechnological industries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An obligately aerobic extremely halophilic alkalithermophilic archaeon, strain JW/NM-HA 15 T , was isolated from the sediments of Wadi An Natrun in Egypt. Phylogenetic analysis based on 16S rRNA and rpoB' gene sequences indicated that it belongs to the family Natrialbaceae of the order Natrialbales. The closest relatives were Natronolimnobius baerhuensis IHC-005 T and Natronolimnobius innermongolicus N-1311 T (95.3 and 94.5 % 16S rRNA gene sequence similarity, respectively). Genome relatedness between strain JW/NM-HA 15 T and its neighbours was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 75.7-85.0, 18.1-20.0, and 70.2-71.0%, respectively. Cells were obligately aerobic, rod-shaped, non-motile, Gram-stain-negative and chemo-organotrophic. The strain grew in the presence of 2.57 M to saturating Na + (optimum 3.25-4.60 M Na + ), at pH 55 °C 7.5-10.5 (optimum pH 55 °C 9.0-9.5), and at 30-56 °C (optimum 52 °C). The major polar lipids consisted of phosphatidylglycerol, methylated phosphatidylglycerolphosphate and two phospholipids. The complete genome size of strain JW/NM-HA 15 T is approximately 3.93 Mb, with a DNA G+C content of 64.1 mol%. On the basis of phylogenetic features, genomic relatedness, phenotypic and chemotaxonomic data, strain JW/NM-HA 15 T was thus considered to represent a novel species within the genus Natronolimnobius, for which the name Natronolimnobius aegyptiacus sp. nov. is proposed. The type strain is JW/NM-HA 15 T (=ATCC BAA-2088 T =DSM 23470 T ).

An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH 55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH 55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Arkashin Schurf (Arkashin) and Zavarzin Spring (Zavarzin), two active thermal pools in the Uzon Caldera, Kamchatka, Russia, were studied for geochemical and microbiological characterization. Arkashin, the smaller of the two pools, had broader temperature and pH ranges, and the sediments had higher concentrations of total As (4,250 mg/kg) relative to Zavarzin (48.9 mg/kg). Glycerol dialkyl glycerol tetraether profiles represented distinct archaeal communities in each pool and agreed well with previous studies of these pools. Although no archaeal 16S rRNA sequences were recovered from Arkashin, sequences recovered from Zavarzin were mostly representatives of the Crenarchaeota and "Korarchaeota," and 13% of the sequences were unclassifiable. The bacterial community in Arkashin was dominated by uncultured "Bacteroidetes," Hydrogenobaculum of the Aquificales and Variovorax of the Betaproteobacteria, and 19% of the sequences remained unclassified. These results were consistent with other studies of As-rich features. The most abundant members of the Zavarzin bacterial community included the Chloroflexi, as well as members of the classes Deltaproteobacteria and Clostridia. In addition, 24% of the sequences were unclassified and at least 5% of those represent new groups among the established Bacterial phyla. Ecological structure in each pool was inferred from taxonomic classifications and bulk stable isotope δ values of C, N, and S. Hydrogenobaculum was responsible for primary production in Arkashin. However, in Zavarzin, the carbon source appeared to be allochthonous to the identified bacterial community members. Additionally, sequences related to organisms expected to participate in N and S cycles were identified from both pools.

The anthropocentric term "extremophile" was introduced more than 30 years ago to describe any organism capable of living and growing under extreme conditions-i.e., particularly hostile to human and to the majority of the known microorganisms as far as temperature, pH, and salinity parameters are concerned. With the further development of studies on microbial ecology and taxonomy, more "extreme" environments were found and more extremophiles were described. Today, many different extremophiles have been isolated from habitats characterized by hydrostatic pressure, aridity, radiations, elevated temperatures, extreme pH values, high salt concentrations, and high solvent/metal concentrations, and it is well documented that these microorganisms are capable of thriving under extreme conditions better than any other organism living on Earth. Extremophiles have also been investigated as far as the search for life in other planets is concerned and even to evaluate the hypothesis that life on Earth came originally from space. Extremophiles are interesting for basic and applied sciences. Particularly fascinating are their structural and physiological features allowing them to stand extremely selective environmental conditions. These properties are often due to specific biomolecules (DNA, lipids, enzymes, osmolites, etc.) that have been studied for years as novel sources for biotechnological applications. In some cases (DNA polymerase, thermostable enzymes), the search was successful and the final application was achieved, but certainly further exploitations are next to come.

Anaerobes can obtain the entire cell's ATP by glycolysis and remove resulting reducing power by fermentation. There is a delicate balance in redox status to obtain a maximal growth of these cells, and the conditions to change redox fluxes can induce kinds of changes in metabolism. The fundamental knowledge on sensing redox status and coupling redox signals with fermentation pathways is essential for the metabolic engineering to control redox fluxes at the molecular level. A redox sensing protein (RSP) was isolated by DNA affinity chromatography, and corresponding gene was mined from genomic sequences of Thermoanaerobacter spp. The RSP shares up to 41% identity with the regulatory proteins which sense NADH and control the expression of NADH dehydrogenase in aerobic microorganisms. The operator sites for RSP were located in all the operons for ethanol fermentation rather than in that of NADH dehydrogenase. The typical operator was identified as a palindromic sequence, -ATTGTTANNNNNNTAACAAT-. NADH caused a transition of RSP from an α-helix rich to β-sheet rich conformation. In an in vitro transcription system of T. ethanolicus, RSP repressed the transcription of an alcohol dehydrogenase, whereas the repression was reversed by adding NADH. Base substitutes in the repeats of the palindrome reduced the affinity between RSP and the operator, and thus delicate regulation could be achieved. This study reveals for the first time a repressor/operator system that couples a redox signal with a fermentation pathway, and the results presented here provide valuable insights for the design of metabolic engineering., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Archaeal microorganisms that grow optimally at Na(+) concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na(+) concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na(+) (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH(opt) of approximately 7, or strongly alkaliphilic, with pH(opt) at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius 'aegyptiacus' (55°C).

The 1,914-bp open reading frame of xylC from Thermoanaerobacterium saccharolyticum JW/SL-YS485 encodes a calculated 73-kDa β-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third β-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression of xylC(m) is due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylC(rec)). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylC(rec) exhibited maximum activity at 65°C and pH(65°C) 6.0, a 1-h half-life at 67°C, a K(m) for p-nitrophenyl-β-D-xyloside of 28 mM, and a V(max) of 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.

Strain JW/YJL-B18(T), a spore-forming, sulfate-reducing bacterium, was isolated from constructed wetland sediment. Cells were curved rods, 0.7-1.2 mum in diameter and 3-7 mum long. Despite being phylogenetically a member of the Gram-type-positive phylum Firmicutes, cells stained Gram-negative at all growth phases. Strain JW/YJL-B18(T) grew at 8-39 degrees C, with an optimum at 32-35 degrees C and no growth at 4 degrees C or below or at 42 degrees C or above. The pH(25 degrees C) range for growth was 5.7-8.2, with an optimum at pH(25 degrees C) 7.0-7.3, and no growth was detected at or below pH 5.2 or at or above pH 8.4. The salinity range for growth was 0-3 % (NaCl/KCl 9 : 1). Strain JW/YJL-B18(T) utilized as carbon and energy sources beef extract, yeast extract, formate, succinate, lactate, pyruvate, ethanol and toluene. Fumarate, sulfate, sulfite and thiosulfate were reduced in the presence of lactate. Arsenate (V) was not used as an electron acceptor. Strain JW/YJL-B18(T) showed no indication of growth under autotrophic conditions. The predominant cellular fatty acids were C(16 : 1) and C(16 : 0). The genomic DNA G+C content was 36.6 mol% (HPLC). 16S rRNA gene sequence analysis indicated that strain JW/YJL-B18(T) fell into the genus Desulfosporosinus, with Desulfosporosinus auripigmenti OREX-4(T) as its closest neighbour with a validly published name (97.9 % similarity). Based on molecular genetic evidence and physiological and biochemical characters including differences in the DNA G+C content, we propose to place strain JW/YJL-B18(T) (=DSM 17734(T) =ATCC BAA-1261(T)) as the type strain of a novel species, Desulfosporosinus youngiae sp. nov.

An anaerobic thermophilic bacterium, designated strain JW/SA-NV4(T), was isolated from a xylan-supplemented enrichment culture from Trego hot spring located within the Black Rock Desert (NV, USA). Cells were generally straight or slightly bent rod-shaped, 0.4-0.8 microm in width and 3-6 microm in length during exponential growth. Cells from stationary phase were variable in size and shape, showing curved or bent morphology. Motility was not seen and flagella were not observed in electron micrographs. Sporulation was not observed. Strain JW/SA-NV4(T) stained Gram-negative but is phylogenetically Gram-type positive. Growth occurred at pH(25 degrees C) 6.8-8.8, with optimum growth at pH 8.4; no growth occurred at pH 9.0 or above or at 6.5 or below. With glucose or xylose as the carbon source, strain JW/SA-NV4(T) grew at 44-74 degrees C; no growth occurred at 76 degrees C or above or at 42 degrees C or below. However, the optimum temperature was 62 and 66 degrees C when grown on glucose and xylose, respectively. The shortest doubling time observed with glucose was approximately 4 h, and with xylose approximately 3.4 h. Strain JW/SA-NV4(T) tolerated an atmosphere containing up to 0.1 % O(2); no growth occurred at a gas atmosphere of 0.2 % O(2). Chemo-organotrophic growth occurred with xylose, glucose, mannose, xylan, pyruvate, fructose, ribose, Casamino acids, mannitol, tryptone, peptone, cellobiose and yeast extract. When grown in mineral media containing 1 g yeast extract l(-1) as an electron donor, thiosulfate and sulfur were reduced to sulfide. The G+C content of the DNA was 38.6 mol% (HPLC). 16S rRNA gene sequence analysis placed strain JW/SA-NV4(T) within the order Thermoanaerobacterales and within the Thermoanaerobacterales Incertae Sedis Family III, specifically between taxa classified within the genera Thermosediminibacter and Thermovenabulum. The closest phylogenetic neighbours were Thermosediminibacter oceani JW/IW-1228P(T) (94.2 % 16S rRNA gene sequence similarity) and Thermosediminibacter litoriperuensis JW/YJL-1230-7/2(T) (94.0 %) [Lee, Y.-J., Wagner, I. D., Brice, M. E., Kevbrin, V. V., Mills, G. L., Romanek, C. S. & Wiegel, J. (2005). Extremophiles 9, 375-383]. Based on physiological and genotypic characteristics, strain JW/SA-NV4(T) (=DSM 18802(T)=ATCC BAA-1454(T)) is proposed to represent the type strain of a novel species in a novel genus, Caldanaerovirga acetigignens gen. nov., sp. nov.

LipA and LipB of Thermosyntropha lipolytica DSM 11003 as previously published are the most alkalithermophilic (pH(opt) (25 degrees C) = 9.4-9.6, T(opt) = 96 degrees C) and thermostable (T (1/2) (24 h) = 74-76 degrees C) lipases currently known. The purified enzymes were analyzed in organic solvents for their ability to catalyze synthesis of diacylglycerols and various alcohol fatty acids. To obtain 100% recovery and avoid a 40% and 50% loss of catalytic activity during lyophilization of purified LipA and LipB, respectively, addition of 1 mg/ml bovine serum albumin (BSA) and 25% polyethylene glycol (PEG400) was required. LipA and LipB catalyzed esterification of fatty acids and alcohols with the highest yields for octyl oleate (LipA) and lauryl oleate (LipB) and also catalyzed synthesis of 1,3-dioleoyl glycerol, 1-oleoyl-3-lauroyl glycerol, and 1-oleoyl-3-octoyl glycerol. Isooctane was the most efficient solvent for esterification reactions at 85 degrees C. Similar to the positional specificity for the hydrolytic reaction in aqueous solutions, LipA and LipB catalyzed in organic solvents the synthesis of diacylglycerol with esterification of position 1 and 3 with a yield of 62% for di-oleoyl glycerol. The reported conversion rates do not represent the full potential of these enzymes, since only 1/100th-1/1,000th of the protein concentrations usually used in commercial processes were available. However, use of slightly increased protein concentrations confirmed the trend to higher yields with higher protein concentrations. The obtained specificity and variety of the reactions catalyzed by LipA and LipB, and their high thermostability allowing synthesis to occur at 90 degrees C, demonstrate their great potentials for industrial applications, particularly in structured lipid biosynthesis for substrates that are less soluble at mesobiotic temperatures.

Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na(+), pH(55 degrees C) 9.5 and 53 degrees C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na(+)(K(+))/H(+) antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8-10.0) and Na(+) concentrations for activity (K(0.5) values 1.0-4.4 mM), properties that correlate with intracellular conditions of N. thermophilus. As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na(+) concentrations and protect biomolecules under multiple extreme conditions.

Novel strains of anaerobic, halophilic, alkalithermophilic bacteria were isolated from sediments of the alkaline, hypersaline lakes of the Wadi An Natrun, Egypt. Strains JW/NM-WN-LUT (and related strains) and JW/NM-WN-LH1T were non-spore-forming, non-motile bacteria with a Gram-type positive cell wall. Optimal growth of strain JW/NM-WN-LUT occurred at pH55 degrees C 9.5, 52 degrees C and with 3.7 M Na+ (2.2 M added NaCl). Optimal growth of strain JW/NM-WN-LH1T occurred at pH55 degrees C 9.9, 51 degrees C and with 3.9 M Na+ (2.3 M added NaCl). Both strains were obligately anaerobic and chemoorganotrophic; producing lactate and acetate as organic acids from pyruvate in different ratios. The DNA G+C contents of strains JW/NM-WN-LUT and JW/NM-WN-LH1T were 41.0 and 42.0 mol%, respectively. The main cellular fatty acids in both strains were anteiso-branched 15:0 and iso-branched 15:0. Phylogenetic analysis showed that both novel isolates belonged to the family Natranaerobiaceae within the order Natranaerobiales. Based on genotypic and phenotypic data, strain JW/NM-WN-LUT (=DSM 18760T=ATCC BAA-1443T) represents the type strain of a novel species, Natranaerobius trueperi sp. nov. Strain JW/NM-WN-LH1T represents a novel genus and species within the family Natranaerobiaceae, Natronovirga wadinatrunensis gen. nov., sp. nov. The type strain of the type species is JW/NM-WN-LH1T (=DSM 18770T=ATCC BAA-1444T).

An NAD(P)H-dependent oxidoreductase has been purified approximately 40-fold from the soluble protein fraction of the dissimilatory iron-reducing, anaerobic, thermophilic bacterium Carboxydothermus ferrireducens. The enzyme, a flavoprotein, has broad-substrate specificity-reducing Fe(3+), Cr(6+), and AQDS with rates of 0.31, 0.33, and 3.3 U mg(-1) protein and calculated NADH oxidation turnover numbers of 0.25, 0.25, and 2.5 s(-1), respectively. Numerous quinones are reduced via a two-electron transfer from NAD(P)H to quinone, thus participating in managing oxidative stress by avoiding the formation of semiquinone radicals.

Several strains of heterotrophic, anaerobic thermophilic bacteria were isolated from hot springs of the Uzon Caldera, Kamchatka, Far East Russia. Strain JW/IW010(T) was isolated from a hot spring within the West sector of the Eastern Thermal field, near Pulsating Spring in the Winding Creek area. Cells of strain JW/IW010(T) were straight to slightly curved rods, 0.5 mum in width and variable in length from 2 to 5 mum and occasionally up to 15 mum, and formed oval subterminal spores. Cells stained Gram-negative, but were Gram-type positive. Growth was observed between 32.5 and 69 degrees C with an optimum around 61 degrees C (no growth occurred at or below 30 degrees C, or at or above 72 degrees C). The pH(60 degrees C) range for growth was 4.2-8.9 with an optimum at 7.1 (no growth occurred at or below pH(60 degrees C) 3.9, or at 9.2 or above). The shortest observed doubling-time at pH(60 degrees C) 6.9 and 61 degrees C was 30 min. Strain JW/IW010(T) was chemo-organotrophic; yeast extract, peptone, Casamino acids and tryptone supported growth. Yeast extract was necessary for the utilization of non-proteinaceous substrates, and growth was observed with inulin, cellobiose, maltose, sucrose, glucose, fructose, galactose, mannose, xylose, trehalose, mannitol, pyruvate and crotonate. The G+C content of the genomic DNA of strain JW/IW010(T) was 33.6 mol% (HPLC method). The major phospholipid fatty acids were iso-15 : 0 (53.5 %), 15 : 0 (11.8 %), 16 : 0 (7.3 %), 10-methyl 16 : 0 (7.3 %) and anteiso-15 : 0 (5.3 %). 16S rRNA gene sequence analysis placed strain JW/IW010(T) in the genus Thermoanaerobacter of the family 'Thermoanaerobacteriaceae' (Firmicutes), with Thermoanaerobacter sulfurigignens JW/SL-NZ826(T) (97 % 16S rRNA gene sequence similarity) and Thermoanaerobacter kivui DSM 2030(T) (94.5 %) as the closest phylogenetic relatives with validly published names. The level of DNA-DNA relatedness between strain JW/IW010(T) and Thermoanaerobacter sulfurigignens JW/SL-NZ826(T) was 64 %. Based on the physiological, phylogenetic and genotypic data, strain JW/IW010(T) represents a novel taxon, for which the name Thermoanaerobacter uzonensis sp. nov. is proposed. The type strain is JW/IW010(T) (=ATCC BAA-1464(T)=DSM 18761(T)). The effectively published strain, 1501/60, of 'Clostridium uzonii' [Krivenko, V. V., Vadachloriya, R. M., Chermykh, N. A., Mityushina, L. L. & Krasilnikova, E. N. (1990). Microbiology (English translation of Mikrobiologiia) 59, 741-748] had approximately 88.0 % DNA-DNA relatedness with strain JW/IW010(T) and was included in the novel taxon.

Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86 degrees C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) gene and transcript sequences, representing a total of 41 amoA operational taxonomic units (OTUs) at 2% identity. The amoA gene sequences were highly diverse, yet they clustered within two major clades of archaeal amoA sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few amoA OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A amoA genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammonia-oxidizing archaea (AOA). The amoA genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring amoA genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that amoA genes were transcribed in situ in one spring and the transcripts were closely related to the amoA genes amplified from the same spring. Our study demonstrates the global occurrence of putative archaeal amoA genes in a wide variety of terrestrial hot springs and suggests that geography may play an important role in selecting different assemblages of AOA.

A novel obligately anaerobic, alkalithermophilic, chemo-organotrophic bacterium was isolated from a small and very shallow geothermally heated pool at Pushino (Kamchatka, Far East Russia). The bacterium, designated strain JW/VK-KS5Y(T), was a Gram staining negative, Gram type positive rod. The cells were sometimes branched, with a tendency to grow in long chains, and were non-sporulating and non-motile. The shortest observed doubling time was 28 min when the novel strain was grown at 54-60 degrees C in 120 mM sodium carbonate-containing medium at pH(25 degrees C) 8.5-9.0. The novel bacterium grew on yeast extract and soytone as sole carbon and energy sources but could also use fumarate, thiosulfate and sulfur as electron acceptors. The DNA G+C content was 32.5 mol%. Based on phylogenetic, DNA-DNA hybridization and phenotypic data, it was concluded that isolate JW/VK-KS5Y(T) (=VKM B-2436(T)=DSM 18970(T)) represents the type strain of a novel species, Anaerobranca zavarzinii sp. nov.

Strain JW/WZ-YB58T, a thermophilic (42-64 degrees C), aerobic, alkalitolerant (pH25 degrees C 6.4-9.7), heterotrophic, sporulating, retarded-peritrichously flagellated and slightly curved rod-shaped bacterium, was isolated from the hot spring Zarvarzin II in the East Thermal Field of the Uzon Caldera, Kamchatka (Far East Russia). The isolate tolerated high concentrations of CO. The major membrane phospholipid fatty acids of JW/WZ-YB58T included iso-C15 : 0 (24.5 %), anteiso-C15 : 0 (18.3 %) and iso-C17 : 0 (17.5 %). The G+C content of the genomic DNA is 45 mol% (HPLC method). Based on 16S rRNA gene sequence analysis and physiological properties, isolate JW/WZ-YB58T (=ATCC BAA-1258T =DSM 17740T) is proposed as the type strain of Caldalkalibacillus uzonensis sp. nov. In contrast to the type species Caldalkalibacillus thermarum, a catalase-reaction-positive aerobe, C. uzonensis was catalase-reaction-negative; thus the description of the genus Caldalkalibacillus is emended to include a catalase-reaction-negative species.

Thermophilic anaerobes are Archaea and Bacteria that grow optimally at temperatures of 50 degrees C or higher and do not require the use of O(2) as a terminal electron acceptor for growth. The prokaryotes with this type of physiology are studied for a variety of reasons, including (a) to understand how life can thrive under extreme conditions, (b) for their biotechnological potential, and (c) because anaerobic thermophiles are thought to share characteristics with the early evolutionary life forms on Earth. Over 300 species of thermophilic anaerobes have been described; most have been isolated from thermal environments, but some are from mesobiotic environments, and others are from environments with temperatures below 0 degrees C. In this overview, the authors outline the phylogenetic and physiological diversity of thermophilic anaerobes as currently known. The purpose of this overview is to convey the incredible diversity and breadth of metabolism within this subset of anaerobic microorganisms.

An obligately anaerobic, spore-forming, Gram-type-positive but Gram-staining-negative thermophilic bacterium, strain JW/YJL-F3(T), was isolated from a Fijian hot spring sediment sample. Cells of strain JW/YJL-F3(T) were straight to slightly curved rods, 0.5-1.2 microm in diameter and 1.5-19 microm long. The temperature range for growth was between 40 and 67 degrees C, with an optimum at 60-63 degrees C. The pH(25 degrees C) range for growth was 4.5-8.4 with an optimum of 6.8. The salinity range for growth was 0-0.5 %. Strain JW/YJL-F3(T) utilized a range of substrates including arabinose, cellobiose, galactose, glucose, inulin, lactose, maltose, mannose, raffinose, ribose, trehalose, xylose and yeast extract as carbon and energy sources. The major fermentation end products from glucose were ethanol, acetate and formate. Strain JW/YJL-F3(T) converted thiosulfate to elemental sulfur, producing sulfur globules. The DNA G+C content was 37.6 mol% as determined by HPLC. Phylogenetic analysis using the 16S rRNA gene sequence indicated that the isolate is distantly related to the clade of the genus Thermoanaerobacterium. However, Thermoanaerobacterium polysaccharolyticum (96.7 % similarity to the type strain) and Thermoanaerobacterium zeae were the closest relatives, forming a separate, well-supported clade together with the novel isolate. Because Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae and strain JW/YJL-F3(T) have different features from other Thermoanaerobacterium species, including a higher G+C content and formate production, and are placed distantly from the remaining species of Thermoanaerobacterium (greater than 10 % distance) in the 16S rRNA gene sequence analysis, we propose to place the new isolate JW/YJL-F3(T) and Thermoanaerobacterium polysaccharolyticum and Thermoanaerobacterium zeae into the novel genus Caldanaerobius gen. nov. as Caldanaerobius fijiensis gen. nov., sp. nov. (the type species), Caldanaerobius polysaccharolyticus comb. nov. and Caldanaerobius zeae comb. nov., respectively. The type strain of Caldanaerobius fijiensis is JW/YJL-F3(T) (=ATCC BAA-1278(T) =DSM 17918(T)).

The ability of anaerobic microorganisms to proliferate under extreme conditions is of widespread importance for microbial physiology, remediation, industry, and evolution. The halophilic alkalithermophiles are a novel group of polyextremophiles. Tolerance to alkaline pH, elevated NaCl concentrations, and high temperatures necessitates mechanisms for cytoplasmic pH acidification; permeability control of the cell membrane; and stability of proteins, the cell wall, and other cellular constituents to multiple extreme conditions. Although it is generally assumed that extremophiles growing at more than one extreme combine adaptive mechanisms for each individual extreme, adaptations for individual extremes often counteract each other. However, in alkaline, hypersaline niches heated via intense solar irradiation, culture-independent analyses have revealed the presence of an extensive diversity of aerobic and anaerobic microorganisms belonging to Bacteria and Archaea that survive and grow under multiple harsh conditions. Thus, polyextremophiles must have developed novel adaptive strategies enabling them to grow and proliferate under multiple extreme conditions. The recent isolation of two novel anaerobic, halophilic alkalithermophiles, Natranaerobius thermophilus and Halonatronum saccharophilum, will provide a platform for detailed biochemical, genomic, and proteomic experiments, allowing a greater understanding of the novel adaptive mechanisms undoubtedly employed by polyextremophiles. In this review, we highlight growth characteristics, ecology, and phylogeny of the anaerobic halophilic alkalithermophiles isolated. We also describe the bioenergetic and physiological problems posed by growth at the multiple extreme conditions of alkaline pH, high NaCl concentration, and elevated temperature under anoxic conditions and highlight recent findings and unresolved problems regarding adaptation to multiple extreme conditions.

Bacillus subtilis (ATCC 6051) reversibly decarboxylates vanillate and 4-hydroxybenzoate under both aerobic and anoxic conditions. Thus, we have identified on the basis of gene sequence homology with Sedimentibacter hydroxybenzoicus and Streptomyces sp. strain D7, a putative B. subtilis hydroxybenzoate decarboxylase. The native form of this enzyme is encoded by 3 genes yclBCD (GI Sequence Identification Nos.: 2632649, 2632650, 2632651) that we have renamed during this research as bsdBCD to align with existing nomenclature. The bsdD gene is reported in the database to be 690 bp; however, our sequence analysis revealed that the size of this gene is in fact 228 bp, an observation that results in a shortening of YclD (i.e., BsdD) from 229 to 75 aa. The corresponding bsdBCD genes were cloned into Escherichia coli, and the heterologously expressed enzyme was assayed for activity. The decarboxylase exhibited a narrow substrate range, with only 2 of the tested substrates, vanillate (Kmapp = 4 mmol.L-1) and 4-hydroxybenzoate (Kmapp = ~1 mmol.L-1), being decarboxylated. The recombinant enzyme had properties similar to that of the native enzyme in respect to specific activity, kinetic properties, bidirectional decarboxylase-carboxylase activity, oxygen insensitivity, and substrate specificity.

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96 degrees C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25 degrees C (pH(25 degrees C) optima) were 9.4 and 9.6, respectively. LipA and LipB at 100 degrees C and pH(25 degrees C) 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C(18:1)) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca2+ and one Mn2+ ion per monomeric enzyme unit. The addition of 1 mM MnCl2 to dialyzed enzyme preparations enhanced the activities at 96 degrees C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60 degrees C and 75 degrees C, respectively, by 4 h.

The phylogenetic diversity of the bacterial and archaeal community in the water and sediments of three large lakes of the Wadi An Natrun was investigated using 16S rRNA clone libraries. The bacterial community was diverse: 769 clones formed 345 operational taxonomic units (OTUs) defined at 99% 16S rRNA sequence identity. The bacterial community in both the water and sediments of the lakes was dominated by clones affiliated with the low G + C Gram-type-positive group, alpha-proteobacteria, and Bacteroidetes, (11-39, 11-30, and 10-37% of OTUs observed, respectively), patterns that have been observed in previously described alkaline, athalassohaline systems. However, a relatively high proportion of Firmicutess-related clones in the water of the lakes and alpha-proteobacteria in the sediments was observed. The bacterial community composition of the water and sediment of the same lake and of different lakes was significantly different (p < 0.05). Operational taxonomic units related to the gamma-proteobacteria were more abundant in the sediment of Lake Fazda, whereas the sediment of Lake UmRisha was dominated by members of the delta-proteobacteria. The proportion of gamma-proteobacterial and Bacteroidetes-affiliated OTUs were predominant in the water of Lake UmRisha and differed significantly from other lake waters (chi-squared analysis, p < or = 0.01). The more oxygenated and dilute nature of Lake Hamra was reflected in its microbial community composition, with the abundance of Bacillales sequences in the water, the absence of Halanaerobiales, Clostridiales, and Archaea in the water, and the presence of representatives of more phyla such as the Actinobacteria, Spirochaetes, and Verrucomicrobia. The archaeal community composition appeared less diverse: 589 clones resulted in 198 OTUs defined at 99% 16S rRNA sequence identity, and all sequences fell into the phylum Euryarchaeota. Phylogenetic analysis showed that many of the sequences were distantly related (83-90% 16S rRNA sequence identity) to cultured and uncultured archaea, with many clones forming clusters that branched deeply within the Euryarchaeota. Forty-two and 53% of the bacterial and archaeal clones had less than 90% 16S rRNA sequence identity to previously described sequences. This indicates that the water and sediments of the Wadi An Natrun harbor a unique and novel prokaryotic diversity that is different from what has been described among other alkaline, athalassohaline lakes.

The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature.

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58(T) was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7-0.8 mum in width and 5.5-12 mum in length and produced terminal spherical spores of 1.2-1.6 mum in diameter with the mother cell swelling around 2 mum in diameter (drumstick-type morphology). Cells grew optimally at pH(25 degrees C) 8.2-8.4 and temperature 50-52 degrees C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO(2 )(both with or without H(2)) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, D: -galactose, D: -mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58(T) is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58(T) (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.

A new group of anaerobic thermophilic bacteria was isolated from enrichment cultures obtained from deep sea sediments of Peru Margin collected during Leg 201 of the Ocean Drilling Program. A total of ten isolates were obtained from cores of 1-2 m below seafloor (mbsf) incubated at 60 degrees C: three isolates came from the sediment 426 m below sea level with a surface temperature of 9 degrees C (Site 1227), one from 252 m below sea level with a temperature of 12 degrees C (Site 1228), and six isolates under sulfate-reducing condition from the lower slope of the Peru Trench (Site 1230). Strain JW/IW-1228P from the Site 1228 and strain JW/YJL-1230-7/2 from the Site 1230 were chosen as representatives of the two identified clades. Based on the 16S rDNA sequence analysis, these isolates represent a novel group with Thermovenabulum and Caldanaerobacter as their closest relatives. The temperature range for growth was 52-76 degrees C with an optimum at around 68 degrees C for JW/IW-1228P and 43-76 degrees C with an optimum at around 64 degrees C for JW/YJL-1230-7/2. The pH(25C) range for growth was from 6.3 to 9.3 with an optimum at 7.5 for JW/IW-1228P and from 5 to 9.5 with an optimum at 7.9-8.4 for JW/YJL-1230-7/2. The salinity range for growth was from 0% to 6% (w/v) for JW/IW-1228P and from 0% to 4.5% (w/v) for JW/YJL-1230-7/2. The G+C [corrected] mol% of the genomic DNA was 46.3 +/- 0.7% (n = 4) for Thermosediminibacter oceani [corrected] JW/IW-1228PT [corrected] and 45.2 +/- 0.7 (n = 6) for Thermosediminibacter litoriperuensis [corrected] JW/YJL-1230-7/2T [corrected] DNA-DNA hybridization yielded 52% similarity between the two strains. According to 16S rRNA gene sequence analysis, the isolates are located within the family, Thermoanaerobacteriaceae. Based on their morphological and physiological properties and phylogenetic analysis, it is proposed that strain JW/IW-1228P(T) is placed into a novel taxa, Thermosediminibacter oceani, gen. nov., sp. nov. (DSM 16646(T)=ATCC BAA-1034(T)), and JW/YJL-1230-7/2(T) into Thermosediminibacter litoriperuensis sp. nov. (DSM 16647(T) =ATCC BAA-1035(T)).

Diverse microbial communities and numerous energy-yielding activities occur in deeply buried sediments of the eastern Pacific Ocean. Distributions of metabolic activities often deviate from the standard model. Rates of activities, cell concentrations, and populations of cultured bacteria vary consistently from one subseafloor environment to another. Net rates of major activities principally rely on electron acceptors and electron donors from the photosynthetic surface world. At open-ocean sites, nitrate and oxygen are supplied to the deepest sedimentary communities through the underlying basaltic aquifer. In turn, these sedimentary communities may supply dissolved electron donors and nutrients to the underlying crustal biosphere.