Cultivation of Piezotolerant and Piezophilic Hyperthermophiles with a Newly Developed Constant High Pressure and Temperature Culturing and Monitoring System.

The Earth's microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules.

Simple In-liquid Staining of Microbial Cells for Flow Cytometry Quantification of the Microbial Population in Marine Subseafloor Sediments.

Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM).

Biochemical characterization of mismatch-binding protein MutS1 and nicking endonuclease MutL from a euryarchaeon Methanosaeta thermophila.

In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR.

The GIY-YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures.

In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown.

Archaeal MutS5 tightly binds to Holliday junction similarly to eukaryotic MutSγ.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

Unlabelled: l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other.

Rapid purification and plasticization of D-glutamate-containing poly-γ-glutamate from Japanese fermented soybean food natto.

Poly-γ-glutamate (PGA) is a major component of mucilage derived from natto, a Japanese fermented food made from soybeans, and PGAs obtained under laboratory's conditions contain numerous d-glutamyl residues. Natto foods are thus promising as a source for nutritionally safe d-amino acids present in intact and digested polymers, although there is little information on the stereochemistry of PGA isolated directly from natto. Here, we describe the development of a new process for rapid purification of PGA using alum and determined the D-glutamate content of natto PGA by chiral high-performance liquid chromatographic analysis.

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa.

Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L.

Crystal structure of the ligand-binding form of nanoRNase from Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of proteins.

NanoRNase (Nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft.

Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis.

Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail.

In vivo, in vitro, and x-ray crystallographic analyses suggest the involvement of an uncharacterized triose-phosphate isomerase (TIM) barrel protein in protection against oxidative stress.

Accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. Characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. Our previous transcriptomic analysis suggested that, in Thermus thermophilus HB8, loss of an oxidized DNA-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria.

Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes.

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B(1), which can serve as an ROS scavenger.

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation.

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase.

Molecular mechanisms of the whole DNA repair system: a comparison of bacterial and eukaryotic systems.

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways.

Structure of RecJ exonuclease defines its specificity for single-stranded DNA.

RecJ is a single-stranded DNA (ssDNA)-specific 5'-3' exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn(2+) or Mg(2+) by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure.

Structural basis for different substrate specificities of two ADP-ribose pyrophosphatases from Thermus thermophilus HB8.

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T.

Insights into different dependence of dNTP triphosphohydrolase on metal ion species from intracellular ion concentrations in Thermus thermophilus.

Deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) from Thermus thermophilus HB8 (TTHB8) hydrolyzes wide variety of dNTPs to deoxyribonucleoside and inorganic triphosphate in magnesium-dependent manner. In this paper, we assess the specificity for various metal ions and of the dNTP triphosphohydrolase activity of the dNTPase from TTHB8. Manganese and cobalt ions more effectively induced the activity for dNTPs than magnesium and, unexpectedly, brought about the degradation of single kind of dNTP.

Structure of archaeal glyoxylate reductase from Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate.

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 A crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported.