Fox Cluster determinants for iron biooxidation in the extremely thermoacidophilic Sulfolobaceae.

Within the extremely thermoacidophilic Sulfolobaceae, the capacity to oxidize iron varies considerably. While some species are prolific iron oxidizers (e.g. Metallosphaera sedula), other species do not oxidize iron at all (e.g. Sulfolobus acidocaldarius). Iron oxidation capacity maps to a genomic locus, referred to previously as the 'Fox Cluster', that encodes putative proteins that are mostly unique to the Sulfolobaceae. The role of putative proteins in the Fox Cluster has not been confirmed, but proteomic analysis here of iron-oxidizing membranes from M. sedula indicates that FoxA2 and FoxB (both cytochrome c oxidase-like subunits) and FoxC (CbsA/cytochrome b domain-containing) are essential. Furthermore, comparative genomics (locus organization and gene disruptions) and transcriptomics (polarity effects and differential expression) connect these genomic determinants with disparate iron biooxidation and respiration measurements among Sulfolobaceae species. While numerous homologous proteins can be identified for FoxA in genome databases (COX-like domains are prevalent across all domains of life), few homologues exist for FoxC or for most other Fox Cluster proteins. Phylogenetic reconstructions suggest this locus may have existed in early Sulfolobaceae, while the only other close homologues to the locus appear in the recently discovered candidate phylum Marsarchaota.

Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaiiO6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13-2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O6-methyl-2'-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix-turn-helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (ß)), forming a heterohexameric structure (α3ß3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5-8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme's surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Life in hot acid: a genome-based reassessment of the archaeal order Sulfolobales.

The order Sulfolobales was one of the first named Archaeal lineages, with globally distributed members from terrestrial thermal acid springs (pH < 4; T > 65°C). The Sulfolobales represent broad metabolic capabilities, ranging from lithotrophy, based on inorganic iron and sulfur biotransformations, to autotrophy, to chemoheterotrophy in less acidophilic species. Components of the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle, as well as sulfur oxidation, are nearly universally conserved, although dissimilatory sulfur reduction and disproportionation (Acidianus, Stygiolobus and Sulfurisphaera) and iron oxidation (Acidianus, Metallosphaera, Sulfurisphaera, Sulfuracidifex and Sulfodiicoccus) are limited to fewer lineages. Lithotrophic marker genes appear more often in highly acidophilic lineages. Despite the presence of facultative anaerobes and one confirmed obligate anaerobe, oxidase complexes (fox, sox, dox and a new putative cytochrome bd) are prevalent in many species (even facultative/obligate anaerobes), suggesting a key role for oxygen among the Sulfolobales. The presence of fox genes tracks with a putative antioxidant OsmC family peroxiredoxin, an indicator of oxidative stress derived from mixing reactive metals and oxygen. Extreme acidophily appears to track inversely with heterotrophy but directly with lithotrophy. Recent phylogenetic re-organization efforts are supported by the comparative genomics here, although several changes are proposed, including the expansion of the genus Saccharolobus.

Complete genome sequence of the Sulfodiicoccus acidiphilus strain HS-1T, the first crenarchaeon that lacks polB3, isolated from an acidic hot spring in Ohwaku-dani, Hakone, Japan.

OBJECTIVE: Sulfodiicoccus acidiphilus HS-1T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1T in the order Sulfolobales, we determined and characterized the genome of this strain. RESULTS: The circular genome of S. acidiphilus HS-1T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution.

Increased chalcopyrite bioleaching capabilities of extremely thermoacidophilic Metallosphaera sedula inocula by mixotrophic propagation.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.

Extremely Thermoacidophilic Metallosphaera Species Mediate Mobilization and Oxidation of Vanadium and Molybdenum Oxides.

Certain species from the extremely thermoacidophilic genus Metallosphaera directly oxidize Fe(II) to Fe(III), which in turn catalyzes abiotic solubilization of copper from chalcopyrite to facilitate recovery of this valuable metal. In this process, the redox status of copper does not change as it is mobilized. Metallosphaera species can also catalyze the release of metals from ores with a change in the metal's redox state. For example, Metallosphaera sedula catalyzes the mobilization of uranium from the solid oxide U3O8, concomitant with the generation of soluble U(VI). Here, the mobilization of metals from solid oxides (V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3) was examined for M. sedula and M. prunae at 70°C and pH 2.0. Of these oxides, only V and Mo were solubilized, a process accelerated in the presence of FeCl3 However, it was not clear whether the solubilization and oxidation of these metals could be attributed entirely to an Fe-mediated indirect mechanism. Transcriptomic analysis for growth on molybdenum and vanadium oxides revealed transcriptional patterns not previously observed for growth on other energetic substrates (i.e., iron, chalcopyrite, organic compounds, reduced sulfur compounds, and molecular hydrogen). Of particular interest was the upregulation of Msed_1191, which encodes a Rieske cytochrome b6 fusion protein (Rcbf, referred to here as V/MoxA) that was not transcriptomically responsive during iron biooxidation. These results suggest that direct oxidation of V and Mo occurs, in addition to Fe-mediated oxidation, such that both direct and indirect mechanisms are involved in the mobilization of redox-active metals by Metallosphaera species.IMPORTANCE In order to effectively leverage extremely thermoacidophilic archaea for the microbially based solubilization of solid-phase metal substrates (e.g., sulfides and oxides), understanding the mechanisms by which these archaea solubilize metals is important. Physiological analysis of Metallosphaera species growth in the presence of molybdenum and vanadium oxides revealed an indirect mode of metal mobilization, catalyzed by iron cycling. However, since the mobilized metals exist in more than one oxidation state, they could potentially serve directly as energetic substrates. Transcriptomic response to molybdenum and vanadium oxides provided evidence for new biomolecules participating in direct metal biooxidation. The findings expand the knowledge on the physiological versatility of these extremely thermoacidophilic archaea.

Enhancement of Metallosphaera sedula Bioleaching by Targeted Recombination and Adaptive Laboratory Evolution.

Thermophilic and lithoautotrophic archaea such as Metallosphaera sedula occupy acidic, metal-rich environments and are used in biomining processes. Biotechnological approaches could accelerate these processes and improve metal recovery by biomining organisms, but systems for genetic manipulation in these organisms are currently lacking. To gain a better understanding of the interplay between metal resistance, autotrophy, and lithotrophic metabolism, a genetic system was developed for M. sedula and used to evaluate parameters governing the efficiency of copper bioleaching. Additionally, adaptive laboratory evolution was used to select for naturally evolved M. sedula cell lines with desirable phenotypes for biomining, and these adapted cell lines were shown to have increased bioleaching capacity and efficiency. Genomic methods were used to analyze mutations that led to resistance in the experimentally evolved cell lines, while transcriptomics was used to examine changes in stress-inducible gene expression specific to the environmental conditions.

Structural Insight into Substrate Specificity of 3-Hydroxypropionyl-Coenzyme A Dehydratase from Metallosphaera sedula.

Metallosphaera sedula is a thermoacidophilic autotrophic archaeon known to utilize the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) as carbon fixation pathway. 3-Hydroxypropionyl-CoA dehydratase (3HPCD) is an enzyme involved in the 3-HP/4-HB cycle by converting 3-hydroxypropionyl-CoA to acryloyl-CoA. To elucidate the molecular mechanism of 3HPCD from M. sedula (Ms3HPCD), we determined its crystal structure in complex with Coenzyme A (CoA). Ms3HPCD showed an overall structure and the CoA-binding mode similar to other enoyl-CoA hydratase (ECH) family enzymes. However, compared with the other ECHs, Ms3HPCD has a tightly formed α3 helix near the active site, and bulky aromatic residues are located at the enoyl-group binding site, resulting in the enzyme having an optimal substrate binding site for accepting short-chain 3-hydroxyacyl-CoA as a substrate. Moreover, based on the phylogenetic tree analysis, we propose that the 3HPCD homologues from the phylum Crenarchaeota have an enoyl-group binding pocket similar to that of bacterial short-chain ECHs.

Inorganic Polyphosphate, Exopolyphosphatase, and Pho84-Like Transporters May Be Involved in Copper Resistance in Metallosphaera sedula DSM 5348T.

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeon Metallosphaera sedula contains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPX Msed , Msed_0891) and four presumed pho84-like phosphate transporters (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism in M. sedula DSM 5348T was evaluated. M. sedula DSM 5348T accumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4 shift. PPX Msed was purified, and the enzyme was found to hydrolyze PolyP in vitro. Essential residues for catalysis of PPX Msed were E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore, M. sedula ppx, pho84-like, and copTMA genes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

Sulfurisphaera javensis sp. nov., a hyperthermophilic and acidophilic archaeon isolated from Indonesian hot spring, and reclassification of Sulfolobus tokodaii Suzuki et al. 2002 as Sulfurisphaera tokodaii comb. nov.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KD-1T, was isolated from an acidic hot spring in Indonesia and characterized with the phylogenetically related species Sulfurisphaera ohwakuensis Kurosawa et al. 1998, Sulfolobus tokodaii Suzuki et al., 2002 and Sulfolobus yangmingensis Jan et al. 1999. Cells of KD-1T were irregular cocci with diameters of 0.9-1.3 µm. The strain grew at 60-90 °C (optimum 80-85 °C), pH 2.5-6.0 (optimum pH 3.5-4.0) and 0-1.0 % (w/v) NaCl concentration. KD-1T grew anaerobically in the presence of S0 (headspace: H2/CO2) and FeCl3 (headspace: N2). Under aerobic conditions, chemolithoautotrophic growth occurred on S0, pyrite, K2S4O6, Na2S2O3 and H2. This strain utilized various complex substrates, such as yeast extract, but did not grow on sugars and amino acids as the sole carbon source. The main core lipids were calditoglycerocaldarchaeol and caldarchaeol. The DNA G+C content was 30.6 mol%. Analyses of phylogenetic trees based on 16S rRNA and 23S rRNA genes indicated that KD-1T formed an independent lineage near Sulfurisphaera ohwakuensis TA-1T, Sulfolobus tokodaii 7T and Sulfolobus yangmingensis YM1T. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, KD-1T represents a novel species of the genus Sulfurisphaera Kurosawa et al. 1998, for which the name Sulfurisphaera javensis sp. nov. is proposed. The type strain is KD-1T (=JCM 32117T=InaCC Ar81T). Based on the data, we also propose the reclassification of Sulfolobus tokodaii Suzuki et al., 2002 as Sulfurisphaera tokodaii comb. nov. (type strain 7T=JCM 10545T=DSM 16993T).

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA-assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1-fold higher than that of the S. solfataricus PCNA-assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.

Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile Metallosphaera sedula.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

Sulfodiicoccus acidiphilus gen. nov., sp. nov., a sulfur-inhibited thermoacidophilic archaeon belonging to the order Sulfolobales isolated from a terrestrial acidic hot spring.

A novel thermoacidophilic archaeon, strain HS-1T, was isolated from the Hakone Ohwaku-dani hot spring in Japan. Cells of strain HS-1T in exponential phase were cocci to irregular cocci with a diameter of 0.8-1.5 µm. The strain grew within a temperature range of 50-70 °C (optimal: 65-70 °C), a pH range of pH 1.4-5.5 (optimal: pH 3.0-3.5) and a NaCl concentration range of 0-2.5 % (w/v). The novel strain grew in aerobic conditions but did not grow anaerobically. Moreover, this strain utilized various complex substrates (beef extract, casamino acids, peptone, tryptone and yeast extract) and sugars (arabinose, xylose, galactose, glucose, maltose, sucrose, raffinose and lactose) as sole carbon sources. No chemolithoautotrophic growth occurred on elemental sulfur, pyrite, K2S4O6, Na2S2O3 or FeSO4 . 7H2O; however, growth by the oxidation of hydrogen occurred weakly. The core lipids were calditoglycerocaldarchaeol (CGTE) and caldarchaeol (DGTE). The DNA G+C content of the strain was 52.0 mol%, which was remarkably higher than those of known species of the order Sulfolobales(31-46.2 %). The growth of the strain was significantly inhibited in the presence of elemental sulfur. Analyses of 16S rRNA and 23S rRNA gene sequences showed that HS-1T belonged to the order Sulfolobales; however, it was distantly related to all known species of the order Sulfolobales (less than 89 % sequence similarity). On the basis of these results, we propose the novel genus, Sulfodiicoccus, in the order Sulfolobales (in the family Sulfolobaceae). The type species of the genus is Sulfodiicoccus acidiphilus sp. nov., and the type strain of the species is HS-1T (=JCM 31740T=InaCC Ar79T).

VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae.

When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during 'uranium-shock' implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Increased acid resistance of the archaeon, Metallosphaera sedula by adaptive laboratory evolution.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3- and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the ß-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO2 into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc.

Novel Hydrogenosomes in the Microaerophilic Jakobid Stygiella incarcerata.

Mitochondrion-related organelles (MROs) have arisen independently in a wide range of anaerobic protist lineages. Only a few of these organelles and their functions have been investigated in detail, and most of what is known about MROs comes from studies of parasitic organisms such as the parabasalid Trichomonas vaginalis Here, we describe the MRO of a free-living anaerobic jakobid excavate, Stygiella incarcerata We report an RNAseq-based reconstruction of S. incarcerata's MRO proteome, with an associated biochemical map of the pathways predicted to be present in this organelle. The pyruvate metabolism and oxidative stress response pathways are strikingly similar to those found in the MROs of other anaerobic protists, such as Pygsuia and Trichomonas This elegant example of convergent evolution is suggestive of an anaerobic biochemical 'module' of prokaryotic origins that has been laterally transferred among eukaryotes, enabling them to adapt rapidly to anaerobiosis. We also identified genes corresponding to a variety of mitochondrial processes not found in Trichomonas, including intermembrane space components of the mitochondrial protein import apparatus, and enzymes involved in amino acid metabolism and cardiolipin biosynthesis. In this respect, the MROs of S. incarcerata more closely resemble those of the much more distantly related free-living organisms Pygsuia biforma and Cantina marsupialis, likely reflecting these organisms' shared lifestyle as free-living anaerobes.

Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal "Shock" Reveal Generic and Specific Metal Responses.

UNLABELLED: The extremely thermoacidophilic archaeon Metallosphaera sedula mobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by "shocking" M. sedula with representative metals (Co(2+), Cu(2+), Ni(2+), UO2 (2+), Zn(2+)) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu(2+) (259 ORFs, 106 Cu(2+)-specific ORFs) and Zn(2+) (262 ORFs, 131 Zn(2+)-specific ORFs) triggered the largest responses, followed by UO2 (2+) (187 ORFs, 91 UO2 (2+)-specific ORFs), Ni(2+) (93 ORFs, 25 Ni(2+)-specific ORFs), and Co(2+) (61 ORFs, 1 Co(2+)-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu(2+) (6-fold) but also in response to UO2 (2+) (4-fold) and Zn(2+) (9-fold). Cu(2+) challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu(2+) resistance or issues in sulfur metabolism. The results indicate that M. sedula employs a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions. IMPORTANCE: The mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.