The model archaeon Pyrococcus furiosus grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPR-associated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The P. furiosus genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.
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.
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.
Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [Topt], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B12-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2 fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α2β2) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.
The vital nature of metal uptake and balance in biology is evident in the highly evolved strategies to facilitate metal homeostasis in all three domains of life. Several decades of study on metals and metalloproteins have revealed numerous essential bio-metal functions. Recent advances in mass spectrometry, x-ray scattering/absorption, and proteomics have exposed a much broader usage of metals in biology than expected. Even elements such as uranium, arsenic, and lead are implicated in biological processes as part of an emerging and expansive view of bio-metals. Here we discuss opportunities and challenges for established and newer approaches to study metalloproteins with a focus on technologies that promise to rapidly expand our knowledge of metalloproteins and metal functions in biology.
metallomics; metalloproteomics; metalloproteins; biometals
Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
Copper (Cu) is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.
Copper (Cu) toxicity is a problem of medical, agricultural, and environmental significance. Cu toxicity severely inhibits growth of plant roots significantly affecting their morphology; Cu overload also accounts for some of the most common metal-metabolism abnormalities and neuropsychiatric problems including Wilson's and Menkes diseases. There is a large body of literature on how Cu enters and exits the cell; the kinetic and structural details of Cu translocation between trafficking, sensing, metabolic, and pumping proteins; and phenotypes associated with defects in metalloregulatory and efflux functions. Although the role of metallochaperones in Cu-cytotoxicity has been poorly studied, it has been observed that in animals deletion of metallochaperones results in elevated intracellular Cu levels along with overexpression of the P1-type ATPase efflux pump, ultimately causing malformation with high mortality. These observations are mechanistically explained by a predictive model of the Cu circuit in Halobacterium salinarum, which serves as an excellent model system for Cu trafficking and regulation in organisms with multiple chaperones. Constructed through iterative modeling and experimentation, this model accurately recapitulates known dynamical properties of the Cu circuit and predicts that intracellular Cu-buffering emerges as a consequence of the interplay of paralogous metallochaperones that traffic and allocate Cu to distinct targets.
We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or “pop-out,” of the selected marker. We used this method to construct a “markerless” deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.
We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal mode assignments, we recorded the NRVS of D14C ferredoxin samples with 36S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains: (Ph4P)2[Fe4S4Cl4]. Several distinct regions of NRVS intensity are identified, ranging from `protein' and torsional modes below 100 cm−1, through bending and breathing modes near 150 cm−1, to strong bands from Fe-S stretching modes between 250 cm−1 and ~400 cm−1. The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra.
The 57Fe partial vibrational densities of states (PVDOS) for the oxidized samples were interpreted by normal mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe4S4]2+/1+ redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
ferredoxin; Pyrococcus furiosus; 57Fe; nuclear resonance vibrational spectroscopy; NRVS; density functional theory; DFT; Mössbauer; synchrotron radiation; resonance Raman; normal mode
Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S0): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (ΔpyrF). The growth of all three mutants on maltose was comparable without S0, but in its presence, the growth of MBX1 was greatly impaired while the growth of NSR1 and SIP1 was largely unaffected. In the presence of S0, MBX1 produced little, if any, sulfide but much more acetate (per unit of protein) than the parent strain, demonstrating that MBX plays a critical role in S0 reduction and energy conservation. In contrast, comparable amounts of sulfide and acetate were produced by NSR1 and the parent strain, indicating that NSR is not essential for energy conservation during S0 reduction. Differences in transcriptional responses to S0 in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S0 reduction system. Genes controlled by the S0-responsive regulator SurR were not as highly regulated in MBX1 and NSR1. SIP1 produced the same amount of acetate but more sulfide than the parent strain. That SipA is not essential for growth on S0 indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S0 reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.
Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2) or, if elemental sulfur (S0) is present, hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H) as an electron carrier and a membrane-bound hydrogenase (MBH) that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔmbhL). Hydrogenase activity in cytoplasmic extracts of various strains demonstrate that SHI is responsible for most of the cytoplasmic hydrogenase activity. The ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (in the form of H2) generated during sugar oxidation. Under conditions of limiting sulfur, a small but significant amount of H2 was produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains unknown.
hydrogenase; energy metabolism; sulfur; ferredoxin; Pyrococcus furiosus; thermophile; anaerobe
We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.
We present structural and biochemical evidence for a redox switch in the archaeal transcriptional regulator SurR of Pyrococcus furiosus, a hyperthermophilic anaerobe. P. furiosus produces H2 during fermentation, but undergoes a metabolic shift to produce H2S when elemental sulfur (S0) becomes available. Changes in gene expression occur within minutes of S0 addition, and the majority of these S0-responsive genes are regulatory targets of SurR, a key regulator involved in primary S0 response. SurR was shown in vitro to have dual functionality, activating transcription of some of these genes, notably the hydrogenase operons, and repressing others, including a gene encoding sulfur reductase. This work demonstrates via biochemical and structural evidence that the activity of SurR is modulated by cysteine residues in a CxxC motif that constitute a redox switch. Oxidation of the switch with S0 inhibits sequence-specific DNA binding by SurR, leading to deactivation of genes related to H2 production and derepression of genes involved in S0 metabolism.
SurR; Pyrococcus furiosus; regulatory transcription factor; elemental sulfur response; hydrogen production; redox switch
We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection, data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline, revealing that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high throughput SAXS is an enabling technology that may change the way that structural genomics research is done.
The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.
Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.
Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.
The hyperthermophilic and anaerobic bacterium Thermotoga maritima ferments a wide variety of carbohydrates, producing acetate, CO2, and H2. Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and reduced ferredoxin are generated. The oxidation of these electron carriers must be coupled to H2 production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from T. maritima does not use either reduced ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H2 production. The enzyme oxidizes NADH and ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H2. It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of ferredoxin (midpoint potential, −453 mV) is used to drive the unfavorable oxidation of NADH (E0′ = −320 mV) to produce H2 (E0′ = −420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H2-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H2 production and of anaerobic metabolism in general.
This work describes the identification and characterization of SurR, P. furiosus sulfur (S0) response regulator. SurR was captured from cell extract using promoter DNA of a hydrogenase operon that is down-regulated in the primary response of P. furiosus to S0, as revealed by DNA microarray experiments. SurR was validated as a sequence-specific DNA binding protein, and characterization of the SurR DNA binding motif GTTn3AAC led to the identification of several target genes that contain an extended motif in their promoters. A number of these were validated to contain upstream SurR binding sites. These SurR targets strongly correspond with ORFs and operons both up- and down-regulated in the primary response to S0. In vitro transcription revealed that SurR is an activator for its own gene as well as for two hydrogenase operons whose expression is down-regulated during the primary S0 response; it is also a repressor for two genes up-regulated during the primary S0 response, one of which encodes the primary S0-reducing enzyme NAD(P)H sulfur reductase. Herein we give evidence for the role of SurR in both mediating the primary response to S0 and controlling hydrogen production in P. furiosus.
regulatory transcription factor; archaea; Pyrococcus furiosus; sulfur response; hydrogen production; SurR
The hyperthermophilic archaeon Pyrococcus furiosus grows optimally near 100°C and undergoes a heat shock response at 105°C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA+ ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of in vitro RNA to P. furiosus whole-genome microarrays. Our results confirmed the phr, the hsp20, and the aaa+ ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5′-TTTAnnnACnnnnnGTnAnnAAAA-3′ (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.
“Anaerocellum thermophilum” DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75°C. It uses a variety of polysaccharides, including crystalline cellulose and untreated plant biomass, and has potential utility in biomass conversion. Here we report its complete genome sequence of 2.97 Mb, which is contained within one chromosome and two plasmids (of 8.3 and 3.6 kb). The genome encodes a broad set of cellulolytic enzymes, transporters, and pathways for sugar utilization and compared to those of other saccharolytic, anaerobic thermophiles is most similar to that of Caldicellulosiruptor saccharolyticus DSM 8903.
The iron-sulfur nitroso compound [Fe4S3(NO)7]− is a broad-spectrum antimicrobial agent that has been used for more than 100 years to combat pathogenic anaerobes. Known as Roussin's black salt (RBS), it contains seven moles of nitric oxide, the release of which was always assumed to mediate its cytotoxicity. Using the hyperthermophilic archaeon Pyrococcus furiosus, it is demonstrated through growth studies, membrane analyses, and scanning electron microscopy that nitric oxide does not play a role in RBS toxicity; rather, the mechanism involves membrane disruption leading to cell lysis. Moreover, insoluble elemental sulfur (S0), which is reduced by P. furiosus to hydrogen sulfide, prevents cell lysis by RBS. It is proposed that S0 also directly interacts with the membranes of P. furiosus during its transfer into the cell, ultimately for reduction by a cytosolic NADPH sulfur reductase. RBS is proposed to be a new class of inorganic antimicrobial agent that also has potential use as an inert cell-lysing agent.
The crystal structure of the hypothetical protein PF0899 from P. furiosus has been determined to 1.85 Å resolution.
The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 Å against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R
free of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.
structural genomics; SECSG; Pfu-871755; PF0899; high-throughput structure
A method of identifying the best structural model for a protein of unknown structure from a list of structural candidates using unassigned 15N-1H residual dipolar coupling (RDC) data and probability density profile analysis (PDPA) is described. Ten candidate structures have been obtained for the structural genomics target protein PF2048.1 using ROBETTA. 15N-1H residual dipolar couplings have been measured from NMR spectra of the protein in two alignment media and these data have been analyzed using PDPA to rank the models in terms of their ability to represent the actual structure.
A number of advantages in using this method to characterize a protein structure become apparent. RDCs can easily and rapidly be acquired, and without the need for assignment, the cost and duration of data acquisition is greatly reduced. The approach is quite robust with respect to imprecise and missing data. In the case of PF2048.1, a 79 residue protein, only 58 and 55 of the total RDC data were observed. The method can accelerate structure determination at higher resolution using traditional NMR spectroscopy by providing a starting point for the addition of NOEs and other NMR structural data.
unassigned NMR data; residual dipolar coupling; PDPA; protein structure modeling; powder pattern analysis; structural genomics