Search

Abstract Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

The order Thermoanaerobacterales currently consists of fermentative anaerobic bacteria, including the genus Caldicellulosiruptor. Caldicellulosiruptor are represented by thirteen species; all, but one, have closed genome sequences. Interest in these extreme thermophiles has been motivated not only by their high optimal growth temperatures (≥70°C), but also by their ability to hydrolyze polysaccharides including, for some species, both xylan and microcrystalline cellulose. Caldicellulosiruptor species have been isolated from geographically diverse thermal terrestrial environments located in New Zealand, China, Russia, Iceland and North America. Evidence of their presence in other terrestrial locations is apparent from metagenomic signatures, including volcanic ash in permafrost. Here, phylogeny and taxonomy of the genus Caldicellulosiruptor was re-examined in light of new genome sequences. Based on genome analysis of 15 strains, a new order, Caldicellulosiruptorales, is proposed containing the family Caldicellulosiruptoraceae, consisting of two genera, Caldicellulosiruptor and Anaerocellum. Furthermore, the order Thermoanaerobacterales also was re-assessed, using 91 genome-sequenced strains, and should now include the family Thermoanaerobacteraceae containing the genera Thermoanaerobacter, Thermoanaerobacterium, Caldanaerobacter, the family Caldanaerobiaceae containing the genus Caldanaerobius, and the family Calorimonaceae containing the genus Calorimonas. A main outcome of ANI/AAI analysis indicates the need to reclassify several previously designated species in the Thermoanaerobacterales and Caldicellulosiruptorales by condensing them into strains of single species. Comparative genomics of carbohydrate-active enzyme inventories suggested differentiating phenotypic features, even among strains of the same species, reflecting available nutrients and ecological roles in their native biotopes.

Brevibacillus massiliensis strain phR is an obligately aerobic microbe that was isolated from human feces. Here, we show that it readily takes up tungsten (W), a metal previously associated only with anaerobes. The W is incorporated into an oxidoreductase enzyme (BmWOR) that was purified from native biomass. BmWOR consists of a single 65 kDa subunit and contains a single W-pyranopterin cofactor and a single [4Fe-4S] cluster. It exhibited high aldehyde-oxidizing activity with very high affinities (apparent Km

Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD+ and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP+, cofactor F420, formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

The sulfur-reducing enzyme MBS and the hydrogen-gas evolving MBH are the evolutionary link between the ancestor Mrp antiporter and the mitochondrial respiratory complex I. Here, the authors characterise MBS from the hyperthermophilic archaeon Pyrococcus furiosus, solve its cryo-EM structure and discuss the structural evolution from Mrp to MBH and MBS and to the modern-day complex I.

Abstract Background Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. Results Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R 2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. Conclusions Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.

ABSTRACT Rhodanobacter species dominate in the Oak Ridge Reservation (ORR) subsurface environments contaminated with acids, nitrate, metal radionuclides, and other heavy metals. To uncover the genomic features underlying adaptations to these mixed-waste environments and to guide genetic tool development, we sequenced the whole genomes of eight Rhodanobacter strains isolated from the ORR site. The genome sizes ranged from 3.9 to 4.2 Mb harboring 3,695 to 4,035 protein-coding genes and GC contents approximately 67%. Seven strains were classified as R. denitrificans and one strain, FW510-R12, as R. thiooxydans based on full length 16S rRNA sequences. According to gene annotation, the top two Cluster of Orthologous Groups (COGs) with high pan-genome expansion rates (Pan/Core gene ratio) were “replication, recombination and repair” and “defense mechanisms.” The denitrifying genes had high DNA homologies except the predicted protein structure variances in NosZ. In contrast, heavy metal resistance genes were diverse with between 7 to 34% of them were located in genomic islands, and these results suggested origins from horizontal gene transfer. Analysis of the methylation patterns in four strains revealed the unique 5mC methylation motifs. Most orthologs (78%) had ratios of nonsynonymous to synonymous substitutions (dN/dS) less than one when compared to the type strain 2APBS1, suggesting the prevalence of negative selection. Overall, the results provide evidence for the important roles of horizontal gene transfer and negative selection in genomic adaptation at the contaminated field site. The complex restriction-modification system genes and the unique methylation motifs in Rhodanobacter strains suggest the potential recalcitrance to genetic manipulation. IMPORTANCE Despite the dominance of Rhodanobacter species in the subsurface of the contaminated Oak Ridge Reservation (ORR) site, very little is known about the mechanisms underlying their adaptions to the various stressors present at ORR. Recently, multiple Rhodanobacter strains have been isolated from the ORR groundwater samples from several wells with varying geochemical properties. Using Illumina, PacBio, and Oxford Nanopore sequencing platforms, we obtained the whole genome sequences of eight Rhodanobacter strains. Comparison of the whole genomes demonstrated the genetic diversity, and analysis of the long nanopore reads revealed the heterogeneity of methylation patterns in strains isolated from the same well. Although all strains contained a complete set of denitrifying genes, the predicted tertiary structures of NosZ differed. The sequence comparison results demonstrate the important roles of horizontal gene transfer and negative selection in adaptation. In addition, these strains may be recalcitrant to genetic manipulation due to the complex restriction-modification systems and methylations.

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

Metabolizing lignocellulosic feedstocks to industrial chemicals by microorganisms requires surmounting the recalcitrance caused by lignin. Here, the authors pair transgenic lignin modified poplar lines with engineered Caldicellusiruptor bescii to achieve biomass solubilization and ethanol conversion without pretreatment.

The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor besciiC. bescii

ABSTRACT Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

A nitrate- and metal-contaminated site at the Oak Ridge Reservation (ORR) was previously shown to contain the metal molybdenum (Mo) at picomolar concentrations. This potentially limits microbial nitrate reduction, as Mo is required by the enzyme nitrate reductase, which catalyzes the first step of nitrate removal. Enrichment for anaerobic nitrate-reducing microbes from contaminated sediment at the ORR yielded Bacillus strain EB106-08-02-XG196. This bacterium grows in the presence of multiple metals (Cd, Ni, Cu, Co, Mn, and U) but also exhibits better growth compared to control strains, including Pseudomonas fluorescens N2E2 isolated from a pristine ORR environment under low molybdate concentrations (

Abstract Background Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Results Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. Conclusions This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Summary Ethanol is an important target for the renewable production of liquid transportation fuels. It can be produced biologically from pyruvate, via pyruvate decarboxylase, or from acetyl‐CoA, by alcohol dehydrogenase E (AdhE). Thermophilic bacteria utilize AdhE, which is a bifunctional enzyme that contains both acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Many of these organisms also contain a separate alcohol dehydrogenase (AdhA) that generates ethanol from acetaldehyde, although the role of AdhA in ethanol production is typically not clear. As acetyl‐CoA is a key central metabolite that can be generated from a wide range of substrates, AdhE can serve as a single gene fuel module to produce ethanol through primary metabolic pathways. The focus here is on the hyperthermophilic archaeon Pyrococcus furiosus, which grows by fermenting sugar to acetate, CO2 and H2. Previously, by the heterologous expression of adhA from a thermophilic bacterium, P. furiosus was shown to produce ethanol by a novel mechanism from acetate, mediated by AdhA and the native enzyme aldehyde oxidoreductase (AOR). In this study, the AOR gene was deleted from P. furiosus to evaluate ethanol production directly from acetyl‐CoA by heterologous expression of the adhE gene from eight thermophilic bacteria. Only AdhEs from two Thermoanaerobacter strains showed significant activity in cell‐free extracts of recombinant P. furiosus and supported ethanol production in vivo. In the AOR deletion background, the highest amount of ethanol (estimated 61% theoretical yield) was produced when adhE and adhA from Thermoanaerobacter were co‐expressed.

The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters inPyrococcus furiosus.

ABSTRACT Contamination from anthropogenic activities has significantly impacted Earth’s biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning. IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology.

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI]O42−) or uranyl oxycation (U[VI]O22+). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.

The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α2β2 structures but the genome contains genes encoding three additional α-subunits. The ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α2β2 isoenzymes. The α-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The β-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.

ABSTRACT Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100°C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [Topt] of 78°C) controlled by a “cold shock” promoter that is upregulated when cells are transferred from 98°C to 72°C. At 98°C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72°C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation. IMPORTANCE Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100°C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100°C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter for protein expression used in this study is activated at suboptimal growth temperatures of P. furiosus, it is a powerful genetic tool for protein expression with minimal interference of the host’s metabolism and without the need for chemical inducers.