Isolation and genomic analysis of a type IV pili-independent Thermus thermophilus phage, φMN1 from a Japanese hot spring.

A Thermus thermophilus lytic phage was isolated from a Japanese hot spring using a type IV pili-deficient strain as an indicator host, and designated as φMN1. Electron microscopic (EM) examination revealed that φMN1 had an icosahedral head and a contractile tail, suggesting that φMN1 belonged to Myoviridae. An EM analysis focused on φMN1 adsorption to the Thermus host cell showed that the receptor molecules for the phage were uniformly distributed on the outer surface of the cells.

Pyrroloquinoline quinone disodium salt improves brain function in both younger and older adults.

Brain function is important for a good quality of life. Pyrroloquinoline quinone disodium salt (PQQ) has been proven to improve brain function and cognition in older adults (above 45 years). In this double-blind, placebo-controlled study, we investigated the effects of PQQ on cognitive function in adults aged between 20 and 65 years.

Polyamines produced by an extreme thermophile are essential for cell growth at high temperature.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C.

Mechanical inhibition of isolated V from V/A-ATPase for proton conductance.

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V domain, with proton flow through the V membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V domain, the V domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the V domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the V/A-ATPase and isolated V at near-atomic resolution, respectively.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms.

Cryo EM structure of intact rotary H-ATPase/synthase from Thermus thermophilus.

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V domains.

The ingenious structure of central rotor apparatus in VoV1; key for both complex disassembly and energy coupling between V1 and Vo.

Vacuolar type rotary H+-ATPases (VoV1) couple ATP synthesis/hydrolysis by V1 with proton translocation by Vo via rotation of a central rotor apparatus composed of the V1-DF rotor shaft, a socket-like Vo-C (eukaryotic Vo-d) and the hydrophobic rotor ring. Reconstitution experiments using subcomplexes revealed a weak binding affinity of V1-DF to Vo-C despite the fact that torque needs to be transmitted between V1-DF and Vo-C for the tight energy coupling between V1 and Vo. Mutation of a short helix at the tip of V1-DF caused intramolecular uncoupling of VoV1, suggesting that proper fitting of the short helix of V1-D into the socket of Vo-C is required for tight energy coupling between V1 and Vo.

Imaging live cell in micro-liquid enclosure by X-ray laser diffraction.

Emerging X-ray free-electron lasers with femtosecond pulse duration enable single-shot snapshot imaging almost free from sample damage by outrunning major radiation damage processes. In bioimaging, it is essential to keep the sample close to its natural state. Conventional high-resolution imaging, however, suffers from severe radiation damage that hinders live cell imaging.

Molecular basis of ADP inhibition of vacuolar (V)-type ATPase/synthase.

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E.

Visualization of two distinct states of disassembly in the bacterial V-ATPase from Thermus thermophilus.

V-ATPases are multisubunit, membrane-bound, energy-converting, cellular machines whose assembly and disassembly is innately connected to their activity in vivo. In vitro V-ATPases show a propensity for disassembly that greatly complicates their functional, and, in particular, structural characterization. Direct structural evidence for early stages of their disassembly has not been reported yet.

Genomic and proteomic characterization of the large Myoviridae bacteriophage ϕTMA of the extreme thermophile Thermus thermophilus.

A lytic phage, designated as ϕTMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ϕTMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ϕTMA was 151,483 bp in length, and its organization was essentially same as that of ϕYS40 except that the ϕTMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted ortholog of the protein involved in the initiation of the ϕYS40 genomic DNA synthesis.

Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs.

Transcription profile of Thermus thermophilus CRISPR systems after phage infection.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage PhiYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.

Molecular mechanism of energy conservation in polysulfide respiration.

Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-A resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate.

ATP hydrolysis and synthesis of a rotary motor V-ATPase from Thermus thermophilus.

Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site.

Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus.

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (DeltapH) and the electrical potential differences (DeltaPsi) across membranes. The optimum rate of synthesis reached 85 s(-1), and the H(+)/ATP ratio of 4.

Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase.

H(+)-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays.

Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus.

The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 A resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY.

N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus.

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following.

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments.

Subunit arrangement in V-ATPase from Thermus thermophilus.

The V0V1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V1 part (ABDF) and a proton channel V0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V0V1-ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI.

Rotation of the proteolipid ring in the V-ATPase.

V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M.

Conserved bases in the TPsi C loop of tRNA are determinants for thermophile-specific 2-thiouridylation at position 54.

2-Thioribothymidine (s(2)T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s(2)T54 is known to be responsible for the thermostability of tRNA. The s(2)T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus.

An enzyme with a deep trefoil knot for the active-site architecture.

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified.