MetFish: a Metabolomics Pipeline for Studying Microbial Communities in Chemically Extreme Environments.

Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here, we report a facile method based on in situ chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exometabolomics analysis in sample matrices containing up to 2 M total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and it can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5 to 6 orders of magnitude) with excellent linearity, and low median interday reproducibility (e.g., 2.6%). MetFish was successfully applied in targeted and untargeted exometabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exometabolome during community succession; in situ in a native prairie soil, whose exometabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exometabolome over time in the well. IMPORTANCE The identification and accurate quantification of metabolites using electrospray ionization-mass spectrometry (ESI-MS) in hypersaline samples is a challenge due to matrix effects. Clean-up and desalting strategies that typically work well for samples with lower salt concentrations are often ineffective in hypersaline samples. To address this gap, we developed and demonstrated a simple yet sensitive and accurate method-MetFish-using chemical derivatization to enable mass spectrometry-based metabolomics in a variety of hypersaline samples from varied ecosystems and containing up to 2 M dissolved salts.

Efficacy of a novel lantibiotic, CMB001, against MRSA.

OBJECTIVES: To evaluate the efficacy of a novel lantibiotic, CMB001, against MRSA biofilms in vitro and in an in vivo experimental model of bacterial infection. METHODS: Antibacterial activity of CMB001 was measured in vitro after its exposure to whole blood or to platelet-poor plasma. In vitro efficacy of CMB001 against a Staphylococcus aureus biofilm was studied using scanning electron microscopy. The maximum tolerable dose in mice was determined and a preliminary pharmacokinetic analysis for CMB001 was performed in mice. In vivo efficacy was evaluated in a neutropenic mouse thigh model of infection. RESULTS: CMB001 maintained its antibacterial activity in the presence of blood or plasma for up to 24 h at 37°C. CMB001 efficiently killed S. aureus within the biofilm by causing significant damage to the bacterial cell wall. The maximum tolerable dose in mice was established to be 10 mg/kg and could be increased to 30 mg/kg in mice pretreated with antihistamines. In neutropenic mice infected with MRSA, treatment with CMB001 reduced the bacterial burden with an efficacy equivalent to that of vancomycin. CONCLUSIONS: CMB001 offers potential as an alternative treatment option to combat MRSA. It will be of interest to evaluate the in vivo efficacy of CMB001 against infections caused by other pathogens, including Clostridioides difficile and Acinetobacter baumannii, and to expand its pharmacokinetic/pharmacodynamic parameters and safety profile.

Isolation, Characterization and Structure Elucidation of a Novel Lantibiotic From Paenibacillus sp.

We have isolated and characterized a novel antibacterial peptide, CMB001, following an extensive screening effort of bacterial species isolated from diverse environmental sources. The bacterium that produces CMB001 is characterized as a Gram (+) bacillus sharing approximately 98.9% 16S rRNA sequence homology with its closest match, Paenibacillus kyungheensis. The molecule has been purified to homogeneity from its cell-free supernatant by a three-step preparative chromatography process. Based on its primary structure, CMB001 shares 81% identity with subtilin and 62% with nisin. CMB001 is active mainly against Gram-positive bacteria and Mycobacteriaceae but it is also active against certain Gram-negative bacteria, including multi-drug resistant Acinetobacter baumannii. It retains full antibacterial activity at neutral pH and displays a low propensity to select for resistance among targeted bacteria. Based on NMR and mass spectrometry, CMB001 forms a unique 3D-structure comprising of a compact backbone with one α-helix and two pseudo-α-helical regions. Screening the structure against the Protein Data Bank (PDB) revealed a partial match with nisin-lipid II (1WCO), but none of the lantibiotics with known structures showed significant structural similarity. Due to its unique structure, resistance profile, relatively broad spectrum and stability under physiological conditions, CMB001 is a promising drug candidate for evaluation in animal models of bacterial infection.

Polyamine-independent growth and biofilm formation, and functional spermidine/spermine N-acetyltransferases in Staphylococcus aureus and Enterococcus faecalis.

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine-free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N-acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine-independent growth and biofilm formation, and presence of functional polyamine N-acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.

Corrigendum: Saliniramus fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from Hot Lake, Washington, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

There was an error in the proposed genus name in the published article, in that the genus 'Salinivirga' was effectively published while this article was in review. Therefore, the genus 'Salinivirga' should be replaced with 'Saliniramus'. For the convenience of future readers, we have included the complete corrected article below, in which all occurrences of the incorrect genus name have been amended: A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Saliniramus fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Saliniramus and Salinarimonas (the type genus of the family).

Salinivirga fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from a photosynthetic mat, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Salinivirga fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Salinivirga and Salinarimonas (the type genus of the family).

Phenotypic responses to interspecies competition and commensalism in a naturally-derived microbial co-culture.

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp. HL-48 and Marinobacter sp. HL-58, to ask two fundamental scientific questions: 1) how do the phenotypes of two naturally co-existing species respond to partnership as compared to axenic growth? and 2) how do growth and molecular phenotypes of these species change with respect to competitive and commensal interactions? We hypothesized - and confirmed - that co-cultivation under glucose as the sole carbon source would result in competitive interactions. Similarly, when glucose was swapped with xylose, the interactions became commensal because Marinobacter HL-58 was supported by metabolites derived from Halomonas HL-48. Each species responded to partnership by changing both its growth and molecular phenotype as assayed via batch growth kinetics and global transcriptomics. These phenotypic responses depended on nutrient availability and so the environment ultimately controlled how they responded to each other. This simplified model community revealed that microbial interactions are context-specific and different environmental conditions dictate how interspecies partnerships will unfold.

Genomic Reconstruction of Carbohydrate Utilization Capacities in Microbial-Mat Derived Consortia.

Two nearly identical unicyanobacterial consortia (UCC) were previously isolated from benthic microbial mats that occur in a heliothermal saline lake in northern Washington State. Carbohydrates are a primary source of carbon and energy for most heterotrophic bacteria. Since CO2 is the only carbon source provided, the cyanobacterium must provide a source of carbon to the heterotrophs. Available genomic sequences for all members of the UCC provide opportunity to investigate the metabolic routes of carbon transfer between autotroph and heterotrophs. Here, we applied a subsystem-based comparative genomics approach to reconstruct carbohydrate utilization pathways and identify glycohydrolytic enzymes, carbohydrate transporters and pathway-specific transcriptional regulators in 17 heterotrophic members of the UCC. The reconstructed metabolic pathways include 800 genes, near a one-fourth of which encode enzymes, transporters and regulators with newly assigned metabolic functions resulting in discovery of novel functional variants of carbohydrate utilization pathways. The in silico analysis revealed the utilization capabilities for 40 carbohydrates and their derivatives. Two Halomonas species demonstrated the largest number of sugar catabolic pathways. Trehalose, sucrose, maltose, glucose, and beta-glucosides are the most commonly utilized saccharides in this community. Reconstructed regulons for global regulators HexR and CceR include central carbohydrate metabolism genes in the members of Gammaproteobacteria and Alphaproteobacteria, respectively. Genomics analyses were supplemented by experimental characterization of metabolic phenotypes in four isolates derived from the consortia. Measurements of isolate growth on the defined medium supplied with individual carbohydrates confirmed most of the predicted catabolic phenotypes. Not all consortia members use carbohydrates and only a few use complex polysaccharides suggesting a hierarchical carbon flow from cyanobacteria to each heterotroph. In summary, the genomics-based identification of carbohydrate utilization capabilities provides a basis for future experimental studies of carbon flow in UCC.

Underlying mechanisms for syntrophic metabolism of essential enzyme cofactors in microbial communities.

Many microorganisms are unable to synthesize essential B vitamin-related enzyme cofactors de novo. The underlying mechanisms by which such microbes survive in multi-species communities are largely unknown. We previously reported the near-complete genome sequence of two ~18-member unicyanobacterial microbial consortia that maintain stable membership on defined medium lacking vitamins. Here we have used genome analysis and growth studies on isolates derived from the consortia to reconstruct pathways for biogenesis of eight essential cofactors and predict cofactor usage and precursor exchange in these communities. Our analyses revealed that all but the two Halomonas and cyanobacterial community members were auxotrophic for at least one cofactor. We also observed a mosaic distribution of salvage routes for a variety of cofactor precursors, including those produced by photolysis. Potentially bidirectional transporters were observed to be preferentially in prototrophs, suggesting a mechanism for controlled precursor release. Furthermore, we found that Halomonas sp. do not require cobalamin nor control its synthesis, supporting the hypothesis that they overproduce and export vitamins. Collectively, these observations suggest that the consortia rely on syntrophic metabolism of cofactors as a survival strategy for optimization of metabolic exchange within a shared pool of micronutrients.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.

Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia.

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set.

Norspermidine is not a self-produced trigger for biofilm disassembly.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Metal resistance and lithoautotrophy in the extreme thermoacidophile Metallosphaera sedula.

Archaea such as Metallosphaera sedula are thermophilic lithoautotrophs that occupy unusually acidic and metal-rich environments. These traits are thought to underlie their industrial importance for bioleaching of base and precious metals. In this study, a genetic approach was taken to investigate the specific relationship between metal resistance and lithoautotrophy during biotransformation of the primary copper ore, chalcopyrite (CuFeS(2)). In this study, a genetic system was developed for M. sedula to investigate parameters that limit bioleaching of chalcopyrite. The functional role of the M. sedula copRTA operon was demonstrated by cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant. Inactivation of the gene encoding the M. sedula copper efflux protein, copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneous M. sedula mutant (CuR1) with elevated metal resistance transformed chalcopyrite at an accelerated rate without affecting chemoheterotrophic growth. Proteomic analysis of CuR1 identified pleiotropic changes, including altered abundance of transport proteins having AAA-ATPase motifs. Addition of the insoluble carbonate mineral witherite (BaCO(3)) further stimulated chalcopyrite lithotrophy, indicating that carbon was a limiting factor. Since both mineral types were actively colonized, enhanced metal leaching may arise from the cooperative exchange of energy and carbon between surface-adhered populations. Genetic approaches provide a new means of improving the efficiency of metal bioleaching by enhancing the mechanistic understanding of thermophilic lithoautotrophy.

Survival of the fittest: overcoming oxidative stress at the extremes of Acid, heat and metal.

The habitat of metal respiring acidothermophilic lithoautotrophs is perhaps the most oxidizing environment yet identified. Geothermal heat, sulfuric acid and transition metals contribute both individually and synergistically under aerobic conditions to create this niche. Sulfuric acid and metals originating from sulfidic ores catalyze oxidative reactions attacking microbial cell surfaces including lipids, proteins and glycosyl groups. Sulfuric acid also promotes hydrocarbon dehydration contributing to the formation of black "burnt" carbon. Oxidative reactions leading to abstraction of electrons is further impacted by heat through an increase in the proportion of reactant molecules with sufficient energy to react. Collectively these factors and particularly those related to metals must be overcome by thermoacidophilic lithoautotrophs in order for them to survive and proliferate. The necessary mechanisms to achieve this goal are largely unknown however mechanistics insights have been gained through genomic studies. This review focuses on the specific role of metals in this extreme environment with an emphasis on resistance mechanisms in Archaea.

Engineering thermoacidophilic archaea using linear DNA recombination.

Thermoacidophilic archaea comprise one of the major classes of extremophiles. Most belong to the family Sulfolobales within the phylum Crenarchaeota. They are of applied interest as sources of hyperstable enzymes, for biomining of base and precious metals, and for evolutionary studies because of their use of eukaryotic-like subcellular mechanisms. Genetic methods are available for several species particularly Sulfolobus solfataricus. This organism has a considerable number of methods available for the construction of novel cell lines with unique functions. This chapter presents recent developments in the use of homologous recombination and linear DNA for the engineering of site-specific changes in the genome of S. solfataricus.

CopR of Sulfolobus solfataricus represents a novel class of archaeal-specific copper-responsive activators of transcription.

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)(-1). Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus.

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

Use of a robust dehydrogenase from an archael hyperthermophile in asymmetric catalysis-dynamic reductive kinetic resolution entry into (S)-profens.

Described is an efficient heterologous expression system for Sulfolobus solfataricus ADH-10 (Alcohol Dehydrogenase isozyme 10) and its use in the dynamic reductive kinetic resolution (DYRKR) of 2-arylpropanal (Profen-type) substrates. Importantly, among the 12 aldehydes tested, a general preference for the (S)-antipode was observed, with high ee's for substrates corresponding to the NSAIDs (nonsteroidal anti-inflammatory drugs) naproxen, ibuprofen, flurbiprofen, ketoprofen, and fenoprofen. To our knowledge, this is the first application of a dehydrogenase from this Sulfolobus hyperthermophile to asymmetric synthesis and the first example of a DYRKR with such an enzyme. The requisite aldehydes are generated by Buchwald-Hartwig-type Pd(0)-mediated alpha-arylation of tert-butyl propionate. This is followed by reduction to the aldehyde in one [lithium diisobutyl tert-butoxyaluminum hydride (LDBBA)] or two steps [LAH/Dess-Martin periodinane]. Treatment of the profenal substrates with SsADH in 5% EtOH/phosphate buffer, pH 9, with catalytic NADH at 80 degrees C leads to efficient DYRKR, with ee's exceeding 90% for 9 aryl side chains, including those of the aforementioned NSAIDs. An in silico model, consistent with the observed broad side chain tolerance, is presented. Importantly, the SsADH-10 enzyme could be conveniently recycled by exploiting the differential solubility of the organic substrate/product at 80 degrees C and at rt. Pleasingly, SsADH-10 could be taken through several "thermal cycles," without erosion of ee, suggesting this as a generalizable approach to enzyme recycling for hyperthermophilic enzymes. Moreover, the robustness of this hyperthermophilic DH, in terms of both catalytic activity and stereochemical fidelity, speaks for greater examination of such archaeal enzymes in asymmetric synthesis.

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism.

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, approximately 2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.