Laser-Induced Skyrmion Writing and Erasing in an Ultrafast Cryo-Lorentz Transmission Electron Microscope.

We demonstrate that light-induced heat pulses of different duration and energy can write Skyrmions in a broad range of temperatures and magnetic field in FeGe. Using a combination of camera-rate and pump-probe cryo-Lorentz transmission electron microscopy, we directly resolve the spatiotemporal evolution of the magnetization ensuing optical excitation. The Skyrmion lattice was found to maintain its structural properties during the laser-induced demagnetization, and its recovery to the initial state happened in the sub-µs to µs range, depending on the cooling rate of the system.

Simultaneous observation of the quantization and the interference pattern of a plasmonic near-field.

Surface plasmon polaritons can confine electromagnetic fields in subwavelength spaces and are of interest for photonics, optical data storage devices and biosensing applications. In analogy to photons, they exhibit wave-particle duality, whose different aspects have recently been observed in separate tailored experiments. Here we demonstrate the ability of ultrafast transmission electron microscopy to simultaneously image both the spatial interference and the quantization of such confined plasmonic fields. Our experiments are accomplished by spatiotemporally overlapping electron and light pulses on a single nanowire suspended on a graphene film. The resulting energy exchange between single electrons and the quanta of the photoinduced near-field is imaged synchronously with its spatial interference pattern. This methodology enables the control and visualization of plasmonic fields at the nanoscale, providing a promising tool for understanding the fundamental properties of confined electromagnetic fields and the development of advanced photonic circuits.

Characterization of salt tolerance in ectoine-transformed tobacco plants (Nicotiana tabaccum): photosynthesis, osmotic adjustment, and nitrogen partitioning.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) biosynthetic genes (ect. ABC) from Halomonas elongata were introduced to tobacco plants using an Agrobacterium-mediated gene delivery system. The genes for ectoine biosynthesis were integrated in a stable manner into the tobacco genome and the corresponding transcripts were expressed. The concentration of ectoine under salt-stress conditions was higher in the roots than in leaves. A close relationship was found between stomatal conductance and the amount of transported nitrogen, suggesting that water transport through the xylem in the stem and transpiration may be involved in nitrogen transport to leaves. The data indicate that the turgor values of the ectoine transgenic lines increased with increasing salt concentration. The data revealed two ways in which ectoine enhanced salinity tolerance of tobacco plants. First, ectoine improved the maintenance of root function so that water is taken up consistently and supplied to shoots under saline conditions. Second, ectoine enhanced the nitrogen supply to leaves by increasing transpiration and by protecting Rubisco proteins from deleterious effects of salt, thereby improving the rate of photosynthesis.

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei.

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.

Curing of symbiotic plasmid of Mesorhizobium huakuii subsp. rengei isolated from Astragalus sinicus.

Astragalus sinicus (Chinese Milk vetch), a green manure leguminous plant, harbors Mesorhizobium huakuii subsp. rengei strain B3 in the root nodules. The visualization of symbiotic plasmid of strain B3 showed the presence of one sym plasmid of about 425 kbp. Curing of sym plasmid by temperature and acrydine orange was studied. Growing rhizobial cells at high temperature (37 degrees C) or treating the cells with acrydine orange at 50 mg/l eliminated sym plasmid of M. huakuii strain B3, which was confirmed by sym plasmid visualization and plant infection test of cured strains.

Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase.

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii.

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.

DNA synthesis and fragmentation in bacteroids during Astragalus sinicus root nodule development.

Histo- and cytochemical techniques were used to study the DNA replication and fragmentation patterns in bacteroids formed by Mesorhizobium huakuii subsp. rengei in nodules of Astragalus sinicus. DNA replication was detected by the incorporation of 5-bromo deoxy-uridine. Signals denoting DNA synthesis were observed in plant nuclei within the nodule meristem and in bacteroids near the meristem. The TUNEL (TdT-mediated dUTP nick-end labeling) assay was used to measure DNA fragmentation. In nutrient-depleted 1-mpi (month(s) post inoculation) nodule sections, some bacteroids were in vacuoles, and DNA fragmentation signals were observed only in such bacteroids. In contrast, 1-mpi nodule sections without nutrient depletion showed neither bacteroid localization in vacuoles nor DNA fragmentation signals. The bacteroid translocation into vacuoles upon nutrient starvation might results from autophagy of the plant. In 2-mpi nodule sections, bacteroids with DNA fragmentation signals appeared within the cytoplasm of some nodule cells in the senescence zone.

Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan.

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.

High yield expression and single step purification of human thionein/metallothionein.

Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purification with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd(7)- or Zn(7)-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus.

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis.

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.

Genetic and protein engineering of diagnostic enzymes, cholesterol oxidase and xylitol oxidase.

For a long time, clinical diagnosis has been made mainly using chemical methods. Recently, several excellent substrate-specific enzymes have been developed and these enzymes are used as diagnostic catalysts. Using enzymes, it is possible to assay for a specific substance from specimens of serum or urine without the need for isolation of the substance which simplifies the process and shortens the assay time. Furthermore, the use of enzymatic assay methods for diagnosis has been facilitated by the developments in genetic engineering which made it possible to overproduce enzymes inexpensively. Here, we review the diagnostic enzymes, cholesterol oxidase and xylitol oxidase, which were successfully overproduced in our laboratory. In particular, the catalytic activity and pH and thermal stabilities of cholesterol oxidase were improved.

Heterologous expression of a gene encoding cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters.

To develop systems for the expression of heterologous genes in probiotic strains of Lactobacillus and Propionibacterium, we used Lactobacillus plantarum and Propionibacterium freudenreichii and a modified gene encoding cholesterol oxidase (choA) from Streptomyces sp. to generate working models. The acetyl coenzyme A carboxylase (acc) promoter derived from the acc operon of L. plantarum L137 and a previously constructed shuttle vector, pRN14, were used to construct vectors for the expression of heterologous genes in lactic acid bacteria. The concentration of cholesterol oxidase in recombinant L. plantarum carrying choA fused to the NH2-terminal region of the first open reading frame of the acc operon was 3.6 mU/mg of protein. Using the promoters from Propionibacterium, namely, P4, P8, and P138, which enabled high-level expression of choA in Escherichia coli, and a previously constructed shuttle vector pPK705, we constructed expression vectors for Propionibacterium. In recombinant P. freudenreichii subsp. shermanii IFO12426, the activities of cholesterol oxidase generated under the control of promoters P4, P8, and P138 were 1.6, 4.3, and 7.2 U/mg of protein, respectively. The expression of heterologous genes may facilitate the production of useful proteins in these economically important bacteria.

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

The involvement of a cysteine proteinase in the nodule development in Chinese milk vetch infected with Mesorhizobium huakuii subsp. rengei.

Cys proteinases play important roles in plant cell development and senescence. A cDNA, AsNODf32, obtained by differential screening of a nodule cDNA library of the leguminous plant Chinese milk vetch (Astragalus sinicus), represents a nodule-specific Cys proteinase similar to that reported for the actinorhizal Alnus glutinosa-Flankia symbiosis. A characteristic feature of this proteinase is the presence of a putative vacuolar targetting signal, LQDA, within its propeptide. Expression of the AsNODf32 gene, which was studied on northern blots and in situ hybridization, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that AsNODf32 was expressed in senescent-infected tissue at the base of the nodule, as well as in interzone II-III of the infected nodules. In addition to degrading old nodule tissues and bacteroids, AsNODf32 protein may be required as a component of tissue remodeling during nodule development.

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.

Molecular design of novel metal-binding oligomeric human metallothioneins.

Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apometallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245-255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals.

Origin of Livistona chinensis var. subglobosa (Arecaceae) on the "islet of the gods": Aoshima, Japan.

RAPD analysis was performed to discuss the origin of Livistona chinensis var. subglobosa using samples from eight localities, Iriomotejima, Ishigakijima, Okinawa, Yakushima, Tanegashima, Cape Sata, Tsukishima, and Aoshima, in Japan. Random amplified polymorphic DNA (RAPD) markers were obtained using five random primers and analyzed by the unweighted pair group method arithmetic (UPGMA). Data from Iriomotejima clustered with data from Aoshima, suggesting the possibility that seeds or green woods were carried by the tidal current from the southern fields of Iriomotejima to Aoshima.

Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells.

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) functions as a compatible osmolyte in the moderate halophile Halomonas elongata OUT30018. Ectoine is biosynthesized by three successive enzyme reactions from aspartic beta-semialdehyde. The genes encoding the enzymes involved in the biosynthesis, ectA, ectB, and ectC, encoding L-2,4-diaminobutyric acid acetyltransferase, L-2, 4-diaminobutyric acid transaminase, and L-ectoine synthase, respectively, have been previously cloned. To investigate the function of ectoine as a compatible solute in plant cells, the three genes were individually placed under the control of the cauliflower mosaic virus 35S promoter and introduced together into cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The transgenic BY2 cells accumulated a small quantity of ectoine (14-79 nmol g(-1) fresh weight) and showed increased tolerance to hyperosmotic shock (900 mOsm). Furthermore, the transgenic BY2 cells exhibited a normal growth pattern even under hyperosmotic conditions (up to 530 mOsm), in which the growth of the untransformed BY2 (wild type) cells was obviously delayed. These results suggest that genetically engineered synthesis of ectoine results in the increased hyperosmotic tolerance of cultured tobacco BY2 cells despite the low level of accumulation of the solute.