Search

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 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]

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]