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Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

The Synechococcus sp. PCC7942 strain carrying a missense mutation in the peptide-binding domain of DnaK3, one of the essential dnaK gene products, revealed temperature-sensitive growth. We also isolated suppressor mutants of this strain. One of the suppressors was mapped in the ribosomal protein gene rpl24 (syc1876), which encodes the 50S ribosomal protein L24. Subcellular localization of three DnaK proteins was determined, and the results indicated that a quantity of DnaK3 was dislocated from membrane-bound polysomes when dnaK3 temperature-sensitive mutant was incubated at non-permissive temperatures. Furthermore, we examined the photosystem II reaction center protein D1 and detected a translational intermediate polypeptide in membrane-bound polysome fractions prepared from dnaK3 temperature-sensitive cells grown at high temperature. These characteristic features of DnaK3 localizations and detection of D1 protein intermediate were not observed in the suppressor mutant even at high temperatures.

The crystal structures and magnetic properties of heterocyclic thiazyl radicals and related materials have been examined. TTTA (=1,3,5-trithia-2,4,6-triazapentalenyl) exhibited a first-order phase ...

Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency.

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.

The draft genome sequence of a soil bacterium, Aquabacterium sp. strain NJ1, capable of utilizing both liquid and solid alkanes, was deciphered. This is the first report of an Aquabacterium genome sequence.

We report here the draft genome sequence of Bradyrhizobium japonicum Is-34, which is incompatible with Rj4 genotype soybeans. A candidate gene involved in this incompatibility was found to be present in this genome.

Monometallic derivatives of tetrakis(1,2,5-thiadiazole)porphyrazine, [TTDPzH2], with main group tervalent metal ions having the formulae [TTDPzMX] (TTDPz = tetrakis(1,2,5-thiadiazole)porphyrazinato dianion; M = Al(III), X = Cl-, Br-, OH-; M = Ga(III), X = Cl-, OH-; M = In(III), X = AcO-) were prepared and investigated by single-crystal X-ray analysis and IR and UV-vis spectroscopy as well as cyclic voltammetry and spectroelectrochemistry. The complexes [TTDPzMX] (M = Al(III), X = Cl-, Br-; M = Ga(III), X = Cl-) were obtained by direct autocyclotetramerization of the precursor 3,4-dicyano-1,2,5-thiadiazole in hot quinoline in the presence of MX3 salts (M = Al(III), Ga(III); X = Cl-, Br-) and were hydrolized to form the corresponding hydroxide derivatives, [TTDPzMOH]. The In(III) complex, [TTDPzIn(OAc)], was obtained from the free-base macrocycle [TTDPzH2] with In(OH)(OAc)2 in CH3COOH. A single-crystal X-ray study was made at 173 K on the two isostructural species [TTDPzMCl] (M = Al(III), Ga(III)), which have space group P, with a = 12.470(14), b = 12.464(13), and c = 13.947(12) angstroms, alpha = 70.72(3), beta = 79.76(3), and gamma = 90.06(3) degrees, V = 2009.3(3) angstroms3, and Z = 4 for [TTDPzAlCl] and a = 12.429(3), b = 12.430(3), and c = 13.851(3) angstroms, alpha = 70.663(6), beta = 79.788(8), and gamma = 89.991(9) degrees, V = 1983.3(7) angstroms3, and Z = 4 for [TTDPzGaCl]. Square pyramidal coordination exists about the M(III) centers, with Cl- occupying the apical position (Al-Cl = 2.171(5) and Ga-Cl = 2.193(1) angstroms). Al(III) and Ga(III) are located at distances of 0.416(6) and 0.444(2) angstroms from the center of the N4 system. The molecular packing consists of stacked double layers with internal and external average interlayer distances of 3.2 and 3.3 angstroms, respectively. IR spectra show nu(Al-Cl) at 345 cm(-1) for [TTDPzAlCl], nu(Al-Br) at 330 cm(-1) for [TTDPzAlBr], and nu(Ga-Cl) at 382 cm(-1) for [TTDPzGaCl]. The UV-vis spectra in weakly basic (pyridine, DMF, DMSO) and acidic solvents (CF3COOH, H2SO4) show the typical intense pi --> pi transition bands in the Soret (300-400 nm) and Q-band regions (640-660 nm), the bands evidencing some dependence on the nature of the solvent, particularly in acidic solutions. Cyclic voltammetry, differential pulse voltammetry, and thin-layer spectroelectrochemical measurements in pyridine and dimethylformamide of the species [TTDPzMX] indicate reversible first and second one-electron reductions, whereas additional ill-defined reductions are observed at more negative potentials. The examined species are much easier to reduce than their phthalocyanine or porphyrin analogues as a result of the remarkable electron-attracting properties of the TTDPz macrocycle which contains annulated strongly electron-deficient thiadiazole rings.

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.

During the course of screening for exoprotease-deficient mutants among Bacillus subtilis gene disruptants, a strain showing such a phenotype was identified. The locus responsible for this phenotype was the previously unknown gene ybaL , which we renamed salA. The predicted gene product encoded by salA belongs to the Mrp family, which is widely conserved among archaea, prokaryotes, and eukaryotes. Disruption of salA resulted in a decrease in the expression of a lacZ fusion of the aprE gene encoding the major extracellular alkaline protease. The decrease was recovered by the cloned salA gene on a plasmid, demonstrating that the gene is involved in aprE expression. Determination of the cis -acting region of SalA on the upstream region of aprE , together with epistatic analyses with scoC , abrB , and spo0A mutations that also affect aprE expression, suggested that salA deficiency affects aprE-lacZ expression through the negative regulator ScoC. Northern and reverse transcription-PCR analyses revealed enhanced levels of scoC transcripts in the salA mutant cells in the transition and early stationary phases. Concomitant with these observations, larger amounts of the ScoC protein were detected in the mutant cells by Western analysis. From these results we conclude that SalA negatively regulates scoC expression. It was also found that the expression of a salA-lacZ fusion was increased by salA deficiency, suggesting that salA is autoregulated.

To explore the diverse photoreceptors of cyanobacteria, we isolated Nostoc sp. strain NIES-3756 from soil at Mimomi-Park, Chiba, Japan, and determined its complete genome sequence. The Genome consists of one chromosome and two plasmids (total 6,987,571 bp containing no gaps). The NIES-3756 strain carries 7 phytochrome and 12 cyanobacteriochrome genes, which will facilitate the studies of phytochrome-based bioengineering.

Using the polycyano−polycadmate host that is a negative-charged Cd cyanide complex with a framework structure built with Cd2+ ions and bridging cyanide ligands, we synthesized a clathrate including methyl viologen dication (MV2+) and o-dimethoxybenzene (ODMB) as guests. The color of the clathrate was reddish-brown, and its origin was considered to be a charge transfer (CT) interaction of a CT complex formed with MV2+ as an acceptor and ODMB as a donor in the host. The wavelength at the CT absorption maximum was largely red shifted as compared with that of the CT complex in an acetonitrile solution. The single-crystal X-ray diffraction analysis revealed a π−π stacking structure of the CT complex and a one-dimensional arrangement of the CT complexes in a channel-like cavity of the framework host. In the array of the CT complexes, MV2+ ions are adjacent to each other and their separation is short. The direction of the transition moment of the CT absorption determined from the structural information and the s...

By adopting a facile synthetic strategy, we obtained a microporous redox-active metal-organic framework (MOF), namely, Cu(2,7-AQDC) (2,7-H2AQDC = 2,7-anthraquinonedicarboxylic acid) (1), and utilized it as a cathode active material in lithium batteries. With a voltage window of 4.0-1.7 V, both metal clusters and anthraquinone groups in the ligands exhibited reversible redox activity. The valence change of copper cations was clearly evidenced by in situ XANES analysis. By controlling the voltage window of operation, extremely high recyclability of batteries was achieved, suggesting the framework was robust. This MOF is the first example of a porous material showing independent redox activity on both metal cluster nodes and ligand sites.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH

To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the Δ rpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the Δ rpmH strain. Either the disruption of yhdP , which is thought to be involved in the efflux of Mg 2+ , or overexpression of mgtE , which plays a major role in the import of Mg 2+ , could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg 2+ content was lower in the Δ rpmH cells than in the wild type, and the Mg 2+ content in the Δ rpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg 2+ . These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg 2+ . In addition, the Mg 2+ content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the Δ rplA (L1) and Δ rplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg 2+ content is influenced by the amount of 70S ribosomes.

The DegS-DegU two-component regulatory system regulates many cellular events in Bacillus subtilis. Genes for DegSU constitutes an operon directed by the P1 promoter and downstream degU is autoregulated via the P3 promoter activated by phosphorylated DegU. In the Gram-positive bacteria, Spx plays a major role in the protection system against oxidative stresses as a transcriptional regulator. Spx is a substrate of the ATP-dependent ClpXP protease. It regulates diamide-stress regulon in addition to many genes with unknown functions. We have found that null mutations for clpX and clpP, which encode the subunits for the protease ClpXP, enhanced the DegU level through activation of the P1 promoter. We isolated four suppressors for the clpP-enhancing effect. Whole-genome sequencing of the suppressors revealed that two have a point mutation in spx and the rest have a deletion of spx. The clpP-enhancing effect on degS-lacZ expression was abolished in the spx disruptant. These results show that the degSU operon is a new target of Spx-mediated positive regulation. Furthermore, we found that the P1 promoter was induced by glucose and that this induction was greatly reduced in the spx mutant. These results suggested that Spx-mediated glucose induction at the P1 promoter.

The unicellular red alga Cyanidioschyzon merolae is used as a model organism to investigate the basic architecture of photosynthetic eukaryotes. We established a stable expression system for the green fluorescent protein fused with the phycocyanin-associated rod linker (APCC) protein in C. merolae, which was clearly localized on the plastid. This system should be useful in the genetic engineering of C. merolae.

Here we report the draft genome sequence of strain Q-1, an iodide (I − )-oxidizing heterotrophic bacterium in the class Alphaproteobacteria isolated from natural gas brine water. The genome sequence contained a multicopper oxidase gene probably responsible for iodide oxidation. A photosynthetic gene cluster was found but genes for carbon-fixation were absent.

The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum.

We sequenced the genomic DNA and the transcribed RNA of the ascomycetous budding yeast Saitoella complicata, which belongs to the earliest lineage (Taphrinomycotina) of ascomycetes. We found 3 protein-coding regions similar to Clr6 of Schizosaccharomyces (a member of Taphrinomycotina). Clr6 has a structure similar to that of Rpd3 and Hos2 of Saccharomyces. These proteins belong to the class 1 histone deacetylase (HDAC) family. The phylogenetic tree showed that the Clr6, Hos2, and Rpd3 lineages are separated in fungal HDACs. Basidiomycetes have 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. On the other hand, whereas ascomycetes except for Schizosaccharomyces have the Hos2 and Rpd3 homologs, and lack the Clr6 homolog, Schizosaccharomyces has the Clr6 and Hos2 homologs, and lacks the Rpd3 homolog. Interestingly, Pneumocystis and Saitoella have 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. Thus, these fungi are the first ascomycete found to possess all 3 types. Our findings indicated that Taphrinomycotina has conserved the Clr6 protein, suggesting that the ancestor of Dikarya (ascomycetes and basidiomycetes) had 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. During ascomycete evolution, Pezizomycotina and Saccharomycotina appear to have lost their Clr6 homologs and Schizosaccharomyces to have lost its Rpd3 homolog.

Organic optoelectronic devices are usually driven by the electric field generated from an electrode potential difference or bias voltage. Although poled ferroelectric domains may produce oriented stray fields, few efforts have been made to utilize them for photocurrent generation in organic devices. Here we show that large net fields caused by incomplete screening during ferroelectric polarization, and which can be 'restored' by short voltage pulses, can facilitate exciton dissociation in organic semiconductors. The oriented fields, comparable with that produced by an electrode potential difference (1~10 MV m(-1)), here are found to be responsible for the photocurrent in our devices. A prototype for an organic photodetector driven by such stray fields is demonstrated. The photoresponsivity, without any optimization, can achieve ~0.1 mA W(-1). This study provides a different operation principle for the generation of photocurrent in organic optoelectronic devices. Furthermore, the polarity-tunable photoresponse may lead to new photoresponsive memory devices.

The Bacillus subtilis clpX and clpP genes are the sites of pleiotropic mutations that adversely affect growth on a variety of media and impair developmental processes such as sporulation and competence development. ClpX is necessary for the post-exponential induction of genes that require the sigmaH form of RNA polymerase for transcription. Both ClpX and ClpP are required for the activation of sigmaA-dependent transcription of the srf operon that encodes surfactin synthetase and the regulatory peptide ComS, required for the development of genetic competence. Transcription of srf is activated by the two-component regulatory system ComPA in response to the peptide pheromone, ComX, which mediates cell density-dependent control. A clpX mutant, although able to produce ComX, is unable to respond to the pheromone. A mutant allele of comP, encoding a product whose activity is independent of ComX, is not able to suppress clpX with respect to srf expression, suggesting that ClpXP acts at the level of ComA-dependent activation of srf transcription initiation. Suppressor mutations of clpX (cxs-1 and cxs-2) were isolated in screens for pseudorevertants exhibiting high levels of srf expression and sigmaH-dependent transcription respectively. One mutation, cxs-1, suppressed a clpP null mutation with respect to srf transcription, but did not overcome the block conferred by clpP on competence development and sporulation. Both cxs-1 and cxs-2 mutations map to the region of the rpoA gene encoding the RNA polymerase alpha C-terminal domain (alphaCTD). The reconstruction of the cxs-1 and cxs-2 alleles of rpoA confirmed that these mutations confer the suppressor phenotype. These findings provide further support for the hypothesis that ClpX and ClpP might be intimately associated with transcription initiation in B. subtilis.

It is very interesting to determine whether disturbance of mastication following occlusal dysfunction has an effect on mental stress. Therefore, an experimental occlusal interference was attached, and the effect on dopamine release from the rat prefrontal cortex was examined.The extracellular fluid dopamine volume of the prefrontal cortex was measured by the brain microdialysis method.1. Though experimental occlusal interference during nonfunctional periods was intraoral, it. was found that the metabolism of the prefrontal cortex dopamine did not change.2. Experimental occlusal interference during functional periods, that is, feeding periods with cubed food under occlusal interference increased the prefrontal cortex dopamine volume. It was clarified that experimental occlusal interference strongly impacts the affect of the rat by changing the metabolism of dopamine in the prefrontal cortex.3. Cubed food and powdered food increased the prefrontal cortex dopamine volume in the same way. It was found that there was no difference in transition of the increase in the prefrontal cortex dopamine volume from the difference in the texture of the food.Experimental occlusal interference related to feeding behavior affected dopamine metabolism in the rat prefrontal cortex, and it was clarified that a strong stress reaction was induced by experimental occlusal interference.

Molecular clusters such as the Mn12 cluster (Mn12) can be used as a cathode-active material in Li batteries. X-ray absorption fine structure studies on the cathode materials in Mn12 molecular cluster batteries demonstrated that the charging/discharging processes include four-electron-redox reaction of Mn12 with a significant change in its molecular structure.

The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk , the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1,3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.

Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity (1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK1 had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.

A coordination polymer, [Cu5(piv)5]n (piv = pivalate), was prepared by a thermal decomposition method. The unit cell of the crystal structure consisted of one right-handed helical chain and one left-handed helical chain, which were formed by an alternation of Cu(I) ions and carboxylate (O-C-O) groups. This chain possessed a pseudo-5(3) screw axis; the second-order structure was suggested to be realized by a balance between the cuprophilicity and a steric effect of the tert-butyl groups.

Summary: A 31 141 bp continuous nucleotide sequence in the region from trnl to pNEXT52 in the Bacillus subtilis 168 genome was determined. In the region, there were 22 ORFs, two complete rRNA operons, and five tRNA genes. It was deduced that the function of one of the ORFs was similar to that of a sigma factor belonging to the ECF (extra-cytoplasmic functions) subfamily. The gene cluster feuA, B, C reported previously for other strains of B. subtilis was also found in strain 168 and located in this region.

As a part of the Bacillus subtilis genome sequencing project, we have determined a 25-kb sequence covering the 17°-19° region. This region contains 26 complete open reading frames (ORFs) including the alkA and adaA/B operon, which encode genes for adaptive response to DNA alkylation. A homology search for the newly identified 21 ORFs revealed that 4 of them exhibit a significant similarity to known proteins, e.g., methicillin-resistant Staphylococcus aureus (MRSA) protein homolog, proteins involved in chloramphenicol resistance, glucosamine synthase and an ABC transporter protein. The remaining 17 ORFs did not show any significant sequence similarities to known gene products in the database.

The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3′-5′ exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3′-5′ exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3′-5′ exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria.

The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency.

The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.

Here, we report the draft genome sequences of Porphyromonas crevioricanis JCM 15906 T and Porphyromonas cansulci JCM 13913 T , which were isolated from a canine oral cavity and were recently united under the single species P. crevioricanis . These two genome sequences are very similar, and yet a high degree of genome rearrangements is observed.

Background The Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector. The BGM vector system has several attractive properties, such as a large cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis. In this study, we developed important additions to the genetic modification methods of the BGM vector system. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally. Results Using two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system. Conclusion We offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. In addition, we demonstrate that this system can be used for mouse transgenesis. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene.

Rechargeable molecular-cluster batteries (MCBs) based on the manganese cluster complex [Mn12 O12 (CH3 CH2 C(CH3 )2 COO)16 (H2 O)4 ] ([Mn12]) that exhibited a capacity of approximately 200 A h kg(-1) in the battery voltage range of 4.0 to 2.0 V were developed. In these batteries, the capacity of approximately 100 A h kg(-1) in the range of 4.0-3.0 V is caused by a chemical reduction from [Mn12](0) to [Mn12](8-) , whereas the other half in the range of 3.0-2.0 V cannot be explained by a redox change of the Mn ions. We performed the cyclic voltammetry (CV) and (7) Li solid-state NMR measurements on the Mn12-MCBs to investigate the origin of the capacity below 3.0 V. Pseudo-rectangular-shaped CV curves in the range of 3.0-2.0 V demonstrate the presence of an electrical double-layer (EDL) capacitance in Mn12-MCBs, which corresponds to approximately 100 A h kg(-1) . (7) Li NMR studies suggest that Li ions form an EDL with electrons in carbon black electrodes in the capacitance voltage range. The capacitance effects are not formed by the single-carbon electrodes alone, but appear only in the mixture of Mn12 and the carbon black electrodes. This type of coexistence of capacitance effects and redox reaction in one electrochemical cell is quite unusual and can serve as a new working principle for high-performance energy-storage devices.

Subcellular localization of three DnaK proteins of cyanobacterium Synechococcus sp. PCC7942 was determined. DnaK1 and DnaK2 proteins were detected mainly in the cytosolic fraction. On the other hand, the DnaK3 protein occurred in large amounts in the thylakoid membrane fraction. Furthermore, DnaK3 was found to be located on the surface of the thylakoid membrane on the cytosol side. Subcellular localization of chimeric and truncated DnaK3 proteins was also determined, and it was suggested that the region a.a. 381 to a.a. 597 of DnaK3 protein, which is considered to correspond to the peptide-binding domain, was required for the association with the thylakoid membrane.

We investigate the making of magnetic hollow spheres of Co3O4 (2) and fcc Co (3) with diameter 500 nm and thickness 40 nm, using polystyrene-bead templates. The spheres are characterized by scanning electron microscope, transmission electron microscope, electron diffraction, and x-ray diffraction. Magnetic measurements on 2 reveal a drastic enhancement of remnant magnetization below TN induced by cooling-field, while measurements on 3 indicate a soft ferromagnetism similar to that of bulk fcc Co.

Cyanobacteria exhibit light-dependent cell growth since most of their cellular energy is obtained by photosynthesis. In Synechococcus elongatus PCC 7942, one of the model cyanobacteria, DNA replication depends on photosynthetic electron transport. However, the critical signal for the regulatory mechanism of DNA replication has not been identified. In addition, conservation of this regulatory mechanism has not been investigated among cyanobacteria. To understand this regulatory signal and its dependence on light, we examined the regulation of DNA replication under both light and dark conditions among three model cyanobacteria, S. elongatus PCC 7942, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. Interestingly, DNA replication activity in Synechocystis and Anabaena was retained when cells were transferred to the dark, although it was drastically decreased in S. elongatus. Glycogen metabolism and respiration were higher in Synechocystis and Anabaena than in S. elongatus in the dark. Moreover, DNA replication activity in Synechocystis and Anabaena was reduced to the same level as that in S. elongatus by inhibition of respiratory electron transport after transfer to the dark. These results demonstrate that there is disparity in DNA replication occurring in the dark among cyanobacteria, which is caused by the difference in activity of respiratory electron transport.

Bacteria have developed various motility mechanisms to adapt to a variety of solid surfaces. A rhizosphere isolate, Paenibacillus sp. NAIST15-1, exhibited unusual motility behavior. When spotted onto 1.5% agar media, Paenibacillus sp. formed many colonies, each of which moved around actively at a speed of 3.6 μm/sec. As their density increased, each moving colony began to spiral, finally forming a static round colony. Despite its unusual motility behavior, draft genome sequencing revealed that both the composition and organization of flagellar genes in Paenibacillus sp. were very similar to those in Bacillus subtilis. Disruption of flagellar genes and flagellar stator operons resulted in loss of motility. Paenibacillus sp. showed increased transcription of flagellar genes and hyperflagellation on hard agar media. Thus, increased flagella and their rotation drive Paenibacillus sp. motility. We also identified a large extracellular protein, CmoA, which is conserved only in several Paenibacillus and related species. A cmoA mutant could neither form moving colonies nor move on hard agar media; however, motility was restored by exogenous CmoA. CmoA was located around cells and enveloped cell clusters. Comparison of cellular behavior between the wild type and cmoA mutant indicated that extracellular CmoA is involved in drawing water out of agar media and/or smoothing the cell surface interface. This function of CmoA probably enables Paenibacillus sp. to move on hard agar media., Author Summary Motility is crucial as it enables bacteria to seek out and colonize suitable environments. Here, a new isolate from the rhizosphere, Paenibacillus sp., exhibited unusual motility behavior. When spotted on low wet, hard agar media, the bacterium formed many colonies, each of which moved around actively like an individual organism. The cells in moving colonies had a large number of flagella, which drove colony movement. Newly identified large extracellular protein was essential to form moving colonies on hard agar media. This protein seems to facilitate motility by drawing water out of agar or smoothing the cell surface interface. On encountering a wet environment, the moving colonies disassembled quickly, and individual cells swam in the water layer, suggesting that moving colonies specially form under low wet conditions. The results describe a novel mechanism that explains how Paenibacillus sp. overcomes environmental challenges by moving on solid surfaces.

A possible above-room-temperature molecular multiferroic, triethylmethylammonium tetrabromoferrate(III) (1), has been discovered. Its ferroelectric and magnetic phase transitions take place at almost the same temperature (∼360 K), resulting in strong magnetodielectric (MD) coupling, with a MD ratio of 18% at 0.6 MHz. Interestingly, 1 also undergoes a low-temperature ferroelectric-ferroelectric phase transition with an Aizu notation of 6mmF6 and small magnetic and dielectric anomalies at 171 K.

Extracellular lipase activity from Ralstonia sp. NT80 is induced significantly by fatty alcohols such as stearyl alcohol. We found that when lipase expression was induced by stearyl alcohol, a 14-kDa protein (designated EliA) was produced concomitantly and abundantly in the culture supernatant. Cloning and sequence analysis revealed that EliA shared 30% identity with the protein-like activator protein of Pseudomonas aeruginosa, which facilitates oxidation and assimilation of n-hexadecane. Inactivation of the eliA gene caused a significant reduction in the level of induction of lipase expression by stearyl alcohol. Furthermore, turbidity that was caused by the presence of emulsified stearyl alcohol, an insoluble material, remained in the culture supernatant of the ΔeliA mutant during the late stationary phase, whereas the culture supernatant of the wild type at 72 h was comparatively clear. In contrast, when lipase expression was induced by polyoxyethylene (20) oleyl ether, a soluble material, inactivation of eliA did not affect the extracellular lipase activity greatly. These results strongly indicate that EliA facilitates the induction of lipase expression, presumably by promoting the recognition and/or incorporation of the induction signal that is attributed to stearyl alcohol.