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In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Therinococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/ proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFel-hydrogenases-based on sequence motifs. [ABSTRACT FROM AUTHOR]

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, was isolated from a deep-sea hydrothermal vent area in Papua New Guinea. The strain requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides, amino acids and sugars. Recently, genome sequencing of Thermococcus onnurineus NA1 was completed. In this study, 2-DE/MS–MS analysis of the cytosolic proteome was performed to elucidate the metabolic characterization of Thermococcus onnurineus NA1 at the protein level. Among the 1,136 visualized protein spots, 110 proteins were identified. Enzymes related to metabolic pathways of amino acids utilization, glycolysis, pyruvate conversion, ATP synthesis, and protein synthesis were identified as abundant proteins, highlighting the fact that these are major metabolic pathways in Thermococcus onnurineus NA1. Interestingly, multiple spots of phosphoenolpyruvate synthetase and elongation factor Tu were found on 2D gels generated by truncation at the N-terminus, implicating the cellular regulatory mechanism of this key enzyme by protease degradation. In addition to the proteins involved in metabolic systems, we also identified various proteases and stress-related proteins. The proteomic characterization of abundantly induced proteins using 2-DE/MS–MS enables a better understanding of Thermococcus onnurineus NA1 metabolism. [ABSTRACT FROM AUTHOR]

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene ( yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester. [ABSTRACT FROM AUTHOR]

The insulin and insulin-like growth factor-1 (IGF-1) receptors mediate signaling for energy uptake and growth through insulin receptor substrates (IRSs), which interact with these receptors as well as with downstream effectors. Oxygen is essential not only for ATP production through oxidative phosphorylation but also for many cellular processes, particularly those involved in energy homeostasis. The oxygen tension in vivo is significantly lower than that in the air and can vary widely depending on the tissue as well as on perfusion and oxygen consumption. How oxygen tension affects IRSs and their functions is poorly under- stood. Our findings indicate that transient hypoxia (i% oxygen) leads to caspase-mediated cleavage of IRS-1 without inducing cell death. The IRS-1 protein level rebounds rapidly upon return to normoxia. Protein tyrosine phosphatases (PTPs) appear to be important for the IRS-1 cleavage because tyrosine phosphorylation of the insulin receptor was decreased in hypoxia and IRS-1 cleavage could be blocked either with H2O2 or with vanadate, each of which inhibits PTPs. Activity of Akt, a downstream effector of insulin and IGF-1 signaling that is known to suppress caspase activation, was suppressed in hypoxia. Overexpression of dominant-negative Akt led to IRS-1 cleavage even in normoxia, and overexpression of constitutively active Akt partially suppressed IRS-1 cleavage in hypoxia, suggesting that hypoxia- mediated suppression of Akt may induce caspase-mediated IRS-1 cleavage. In conclusion, our study elucidates a mechanism by which insulin and IGF-1 signaling can be matched to the oxygen level that is available to support growth and energy metabolism. [ABSTRACT FROM AUTHOR]

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-lace forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s20,w 7.0 S; Mapp 169, 200) at concentrations ≤3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a Mapp of 1 ⊗ 106. Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles. [ABSTRACT FROM AUTHOR]

Seeks to determine the effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of Streptomyces albidoflavus SMF301. Production of three types of proteases; Methodology utilized in the study; Results and conclusion of the study.

Identifies three proteases in cultures of Streptomyces albidoflavus SMF301. Micro-organism and media; Strain maintenance and culture conditions; Measurement of submerged spore formation; Assay of proteases; Effect of pH and temperature; Purification of proteases; Chemicals, reagents and reproducibility.

It has been reported that the frhAGB-encoded hydrogenase from Thermococcus onnurineus NA1 is homologous to the F420-reducing hydrogenase from methanogens and can reduce thioredoxin reductase (TrxR) via direct electron transfer from H2 oxidation. In this study, to find other interaction targets of frhAGB-encoded hydrogenase, we searched for structural homologs of TrxR using the Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) protein database search program and AlphaFold Protein Structure Database. Fdh3B (TON_0542), a subunit of the formate dehydrogenase 3 (Fdh3), showed the most similar structure to TrxR in its domains. The interaction potential of Fdh3B with frhAGB-encoded hydrogenase was demonstrated by measuring H2-dependent NADP+ reduction by a mixture of purified proteins. Similarly, the H2-dependent NADP+ -reducing activity of Fdh3 whole complex and two other TrxR homologs encoded by TON_0702 and TON_1376 was determined. It was also demonstrated that the Fdh3 complex could reduce CO2 to formate in the presence of the frhAGB-encoded hydrogenase and H2, as shown for the H2-dependent CO2 reductase (HDCR) of acetogen. The in vivo contribution of the frhAGB-encoded hydrogenase and Fdh3 to H2-dependent CO2 reduction was investigated using a resting cell assay, and formate production was significantly decreased in fdh3A or frhA deletion mutants. In conclusion, the frhAGB-encoded hydrogenase was identified to directly transfer electrons to Fdh3 without an electron carrier, similar to the mechanism observed for TrxR. These results expand our understanding of the functional role of the frhAGB-encoded hydrogenase in non-methanogenic Thermococcus species. IMPORTANCE The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGBencoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3. [ABSTRACT FROM AUTHOR]

Novosphingobium pentaromativorans US6-1 showed a good ability to degrade high-molecular-weight pofycydic aromatic hydrocarbons. We report the draft genome sequence of strain US6-1, which contains a main chromosome (5,096,413 bp, G+C content of 63.1%) and two plasmids (188,476 and 60,085 bp). The majority of the aromatic-hydrocarbon-degrading genes are encoded in the larger plasmid. [ABSTRACT FROM AUTHOR]

Kordia algicida OT-1 is an algicidal bacterium against the bloom-forming microalgae. The genome sequence of K. algicida revealed a number of interesting features, including the degradation of macromolecules, the biosynthesis of carotenoid pigment and secondary metabolites, and the capacity for gliding motility, which might facilitate the understanding of algicidal mechanisms. [ABSTRACT FROM AUTHOR]

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO2, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus. [ABSTRACT FROM AUTHOR]

Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H+ translocation across the cytoplasmic membrane that then drives Na+ translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na+/H+ antiporter module. The electrochemical Na+ gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na+/H+ antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex Hike electron transport chains. [ABSTRACT FROM AUTHOR]

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X1–S–X2–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C4) among various p-nitrophenyl esters (C2 to C18), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K m (307 μM), k cat (5.72 s−1), and k cat/ K m (18.61 s−1 mM−1). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment. [ABSTRACT FROM AUTHOR]

Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed ( R)-styrene oxide, whereas EEH3 preferred to hydrolyze ( S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze ( R)- and ( S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40–55 °C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide. [ABSTRACT FROM AUTHOR]

This study is focused on the involvement of the unusual nucleotide (p)ppGpp during the morphological and physiological differentiation of Streptornyces clavuligerus. In particular, the functional and structural elements of two genes encoding the proteins RelA and Rsh were identified. The relA gene encodes an 843 aa protein (RelA), while the rsh gene encodes a 738 aa protein (Rsh). The relA and rsh genes were disrupted by the insertion of a hygromycin resistance gene and an apramycin resistance gene, respectively. The synthesis of ppGpp in the relA gene-disrupted mutant was completely eliminated under conditions of starvation for amino acids, whereas synthesis persisted, but was greatly reduced in the rsh gene-disrupted mutant. The relA gene-disrupted mutant had a bald appearance on agar plate cultures and retarded growth in submerged culture, while the rsh-disrupted mutant was unchanged in growth characteristics relative to the wild-type culture. The production of both clavulanic acid and cephamycin C were completely abolished in the relA-disrupted mutant. Thus, it is concluded that the relA gene rather than rsh is essential for morphological and physiological differentiation in S. clavuligerus and that RelA primarily governs the stringent response of S. clavuligerus to starvation for amino acids. [ABSTRACT FROM AUTHOR]

To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, "Thermococcus onnurineus" NA1T, were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm-3, were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ρ25]=1.2434 g cm-3) and C (ρ25=1.2648 g cm-3), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1T EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1T were packaged into EVs, except for an∼9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3′-terminal sequence and the absence of the 5′-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves. [ABSTRACT FROM AUTHOR]

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H² when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H²-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (=1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe-S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H²-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments. [ABSTRACT FROM AUTHOR]

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80°C and still retained an activity of 2.5 units/mg of protein at 45°C. The high activity of the enzyme at 45°C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45°C and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea. [ABSTRACT FROM AUTHOR]

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑ strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter-1 h-1) and the specific H2 production rate (249.6 mmol g-1 h-1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production. [ABSTRACT FROM AUTHOR]

Hydrogenogenic CO oxidation (CO + H2O → CO2 + H2) has the potential for H2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MCO1, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na+/H+ antiporter and a 1.8-fold-higher specific activity for CO-dependent H2 production than did the wild-type strain. The H2 production potential of the MCO1 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H2 production rate of the engineered strain was severalfold higher than those of any other COdependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe. [ABSTRACT FROM AUTHOR]

Formate-dependent proton reduction to H2 (HCOO- + H2O → HCO3 + H2) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H2 accumulation to a partial pressure of 1 × 105 to 7 × 105 Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (-1 to -3 kJ mol-1). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as -5 kJ mol-1, which are less than the biological minimum energy quantum, -20 kJ mol-1, as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H2 storage material, the H2 production potential of the strain was assessed. The volumetric H2 production rate increased linearly with increasing cell density, leading to 2,820 mmol liter-1 h-1 at an optical density at 600 nm (OD600) of 18.6, and resulted in the high specific H2 production rates of 404 ± 6 mmol g-1 h-1.The H2 productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H2-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems. [ABSTRACT FROM AUTHOR]

We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature. [ABSTRACT FROM AUTHOR]

It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various βlactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estUl) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-Iactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstUl) was further characterized. EstUl showed esterase activity toward various chromogenicp-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstUl could hydrolyze β-βLactam antibiotics. EstUl was able to hydrolyze firstgeneration β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, arid cefazolin. In a kinetic study, EstUl showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstUl plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstUl in both activities. [ABSTRACT FROM AUTHOR]

Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33-58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30-35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology. [ABSTRACT FROM AUTHOR]

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine-lysine catalytic dyad. We report the 2.0-Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight- and weak-binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP-driven protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions. [ABSTRACT FROM AUTHOR]

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H2 (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol−1) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H2 consumption by the methanogens, which lowers the H2 partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H2. Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H2-producing growth. The actual ΔG values for the formate metabolism are calculated to range between −8 and −20 kJ mol−1 under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability. [ABSTRACT FROM AUTHOR]

The TON_0002 gene, which is in close proximity to the DNA polymerase locus in Thermococcus onnurineus NA1, has been shown to encode an inorganic pyrophosphatase. Its genomic position and function suggest a role for pyrophosphate hydrolysis during DNA polymerization. This is the first report of an inorganic pyrophosphatase belonging to the haloacid dehalogenase superfamily, in which unique residues in motif I and II have been replaced with Trp and Gly, respectively. The optimum pyrophosphatase activity of the recombinant enzyme occurred at pH 6, and it displayed an absolute dependence on divalent metal ions, among which Ni2+ was the most efficient. The site-specific mutation of the Gly residue in motif II to Ala or Ser residue exhibited only a slight change in the enzymatic activity and the Km value. [ABSTRACT FROM AUTHOR]

The article presents a study that describes the cloning of the gene and characterization of the recombinant enzyme with regard to its enzymatic properties. Escherichia coli was used for plasmid propagation and nucleotide sequencing. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-hp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1--APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nϵ-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 mm at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP. [ABSTRACT FROM AUTHOR]

The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON_1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°. [ABSTRACT FROM AUTHOR]

Pyrococcus sp. strain NA2, isolated from a deep-sea hydrothermal vent sample, is a novel marine hyperthermophilic archaeon that grows optimally at 93°C. The complete genome sequence of the strain contains all the genes for the tricarboxylic acid cycle except for succinate dehydrogenase/fumarate reductase, but the genome does not encode proteins involved in polysaccharide utilization. [ABSTRACT FROM AUTHOR]

The complete genome sequence of "Candidatus Puniceispirillum marinum" IMCC1322, the first cultured representative of the SAR116 clade in the Alphaproteobacteria, is reported here. The genome contains genes for proteorhodopsin, aerobic-type carbon monoxide dehydrogenase, dimethylsulfoniopropionate demethylase, and C1 compound metabolism. The genome information proposes the SAR116 group to be metabolic generalists in ocean nutrient cycling. [ABSTRACT FROM AUTHOR]

Two hypothetical genes were functionally verified to be a pyrophosphatase and a PAP phosphatase in Thermococcus onnurineus NA1. This is the first report of the pyrophosphatases and the PAP phosphatases being organized in the gene clusters of the sulfate activation system only in T. onnurineus NA1 and "Pyrococcus abyssi". [ABSTRACT FROM AUTHOR]

The TON_0887 gene was identified as the missing histidinol-phosphate phosphatase (HolPase) in the hyperthermophilic archaeon "Thermococcus onnurineus" NA1. The protein contained conserved motifs of the DDDD superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong HolPase activity. In this study, we functionally assessed for the first time the monofunctional DDDD-type HolPase, which is organized in the gene cluster. [ABSTRACT FROM AUTHOR]