Eco-Benign Orange-Hued Pigment Derived from Aluminum-Enriched Biogenous Iron Oxide Sheaths.

Inorganic pigments have been widely used due to their low cost of production, strong hiding power, and chemical resistance; nevertheless, they have limited hue width and chromaticity. To eliminate these disadvantages, we herein propose the use of an ingenious biotemplate technique to produce Al-enriched biogenic iron oxide (BIOX) materials. Spectrophotometric color analysis showed that high levels of Al inclusion on heat-treated BIOX samples produced heightened yellowish hues and lightness. The Al-enriched BIOX sheaths exhibited a stable tubular structure and excellent thermal stability of color tones after heating at high temperatures and repetitive heat treatments. Ultrastructural analysis and mechanical destruction experiments revealed that the highly chromatic orange-hue of these pigments are ascribed probably to an ingenious cylindrical nanocomposite architecture composed of putative Fe-included low crystalline Al oxide regions and hematite particles embedded therein. The present work therefore demonstrates that the bioengineered material can serve as an epochal orange-hued inorganic pigment with low toxicity and marked thermostability that should meet large industrial demand.

Preparation and Characterization of Additional Metallic Element-Containing Tubular Iron Oxides of Bacterial Origin.

Biogenic microtubular iron oxides (BIOXs) derived from Leptothrix spp. are known as promising multifunctional materials for industrial applications such as ceramic pigments and catalyst carriers. Here, we report unprecedented BIOX products with additive depositions of various metallic elements prepared by a newly devised "two-step" method using an artificial culture system of Leptothrix cholodnii strain OUMS1; the method comprises a biotic formation of immature organic sheaths and subsequent abiotic deposition of Fe and intended elements on the sheaths. Chemical composition ratios of the additional elements Al, Zr, and Ti in the respective BIOXs were arbitrarily controllable depending on initial concentrations of metallic salts added to reaction solutions. Raman spectroscopy exemplified an existence of Fe-O-Al linkage in the Al-containing BIOX matrices. Time-course analyses revealed the underlying physiological mechanism for the BIOX formation. These results indicate that our advanced method can contribute greatly to creations of innovative bioderived materials with improved functionalities.

High-Quality Inorganic Red Pigment Prepared by Aluminum Deposition on Biogenous Iron Oxide Sheaths.

Naturally occurring tubular iron oxides produced by aquatic bacteria in Leptothrix spp. are promising raw materials for hematite-based red pigments because of the higher heat resistance as compared with chemically synthesized hematite compounds. Here, we report iron oxide red pigments prepared through an additive deposition of aluminum on culture-based biogenous iron oxide (cBIOX) sheaths using an artificial culture system of L. cholodnii strain OUMS1. The heat-treated Al-containing cBIOXs exhibited elevated chroma and lightness along with increasing Al contents and enhanced thermal stability of color tones to repetitive heat treatments. XRD analysis showed a monophasic pattern of hematite in the Al-rich cBIOX after heating at a wide range of high temperatures. Micromorphology analyses revealed that putative Al oxide regions present among hematite particles plausibly prevented the grain growth of hematite during heat treatments. The results therefore demonstrate that the bioderived Al-rich iron oxide sheaths can serve as innovative inorganic red pigments feasible for industrial applications.

Amino group in Leptothrix sheath skeleton is responsible for direct deposition of Fe(III) minerals onto the sheaths.

Leptothrix species produce microtubular organic-inorganic materials that encase the bacterial cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with inorganic material. Functional carboxyl groups of polysaccharides in these fibrils are considered to attract and bind metal cations, including Fe(III) and Fe(III)-mineral phases onto the fibrils, but the detailed mechanism remains elusive. Here we show that NH2 of the amino-sugar-enriched exopolymer fibrils is involved in interactions with abiotically generated Fe(III) minerals. NH2-specific staining of L. cholodnii OUMS1 detected a terminal NH2 on its sheath skeleton. Masking NH2 with specific reagents abrogated deposition of Fe(III) minerals onto fibrils. Fe(III) minerals were adsorbed on chitosan and NH2-coated polystyrene beads but not on cellulose and beads coated with an acetamide group. X-ray photoelectron spectroscopy at the N1s edge revealed that the terminal NH2 of OUMS1 sheaths, chitosan and NH2-coated beads binds to Fe(III)-mineral phases, indicating interaction between the Fe(III) minerals and terminal NH2. Thus, the terminal NH2 in the exopolymer fibrils seems critical for Fe encrustation of Leptothrix sheaths. These insights should inform artificial synthesis of highly reactive NH2-rich polymers for use as absorbents, catalysts and so on.

Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cells.

Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aqueous-phase inorganics, especially Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid organic-inorganic nature. To verify our assumption that the organic skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examined the sorption potential of chemically and enzymatically prepared protein-free organic sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% atomic percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform infrared spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5-10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, especially via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.

Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths.

Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amounts of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath production and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examined using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissociation, re-aggregation and re-elongation events are the basis of the rapid, massive production of Leptothrix sheaths typically observed at macroscopic scales.

Abiotic Deposition of Fe Complexes onto Leptothrix Sheaths.

Bacteria classified in species of the genus Leptothrix produce extracellular, microtubular, Fe-encrusted sheaths. The encrustation has been previously linked to bacterial Fe oxidases, which oxidize Fe(II) to Fe(III) and/or active groups of bacterial exopolymers within sheaths to attract and bind aqueous-phase inorganics. When L. cholodnii SP-6 cells were cultured in media amended with high Fe(II) concentrations, Fe(III) precipitates visibly formed immediately after addition of Fe(II) to the medium, suggesting prompt abiotic oxidation of Fe(II) to Fe(III). Intriguingly, these precipitates were deposited onto the sheath surface of bacterial cells as the population was actively growing. When Fe(III) was added to the medium, similar precipitates formed in the medium first and were abiotically deposited onto the sheath surfaces. The precipitates in the Fe(II) medium were composed of assemblies of globular, amorphous particles (ca. 50 nm diameter), while those in the Fe(III) medium were composed of large, aggregated particles (≥3 µm diameter) with a similar amorphous structure. These precipitates also adhered to cell-free sheaths. We thus concluded that direct abiotic deposition of Fe complexes onto the sheath surface occurs independently of cellular activity in liquid media containing Fe salts, although it remains unclear how this deposition is associated with the previously proposed mechanisms (oxidation enzyme- and/or active group of organic components-involved) of Fe encrustation of the Leptothrix sheaths.

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5-phosphatase, is required for proper development of intercalary meristem.

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.

Arabidopsis replication protein A 70a is required for DNA damage response and telomere length homeostasis.

Replication protein A1 (RPA1/RPA70) forms a heterotrimeric complex together with RPA2/RPA32 and RPA3/RPA14 subunits which plays essential roles in various aspects of DNA metabolism including replication, repair, recombination and telomere maintenance. Compared with RPA70 in yeast and mammals, limited information is available about the factor in plants. In this study, we analyzed the functions of AtRPA70a, which is most similar to human RPA70 among four paralogs in Arabidopsis thaliana. RNA blot analysis showed that AtRPA70a is expressed ubiquitously in plant organs containing differentiated and meristematic tissues, while its expression was up-regulated in response to DNA damage stress. Yeast two-hybrid and co-immunoprecipitation analyses showed that AtRPA70a interacted preferentially with Arabidopsis RPA32a, one of two paralogs. Inactivation of AtRPA70a by T-DNA insertion did not affect growth under normal conditions, but resulted in increased sensitivity to genotoxic agents such as methylmethane sulfonate, bleomycin and hydroxyurea. Terminal restriction fragment analysis revealed that telomere lengths in an AtRPA70a-deficient line were significantly larger than in the wild type, whereas those in the mutant expressing antisense AtTERT (telomerase catalytic subunit gene) were shortened during successive generations. These results demonstrate that AtRPA70a is involved in repair of double-strand DNA breaks and possibly contributes to telomerase-dependent telomere length regulation.

Loss of calmodulin binding to Bax inhibitor-1 affects Pseudomonas-mediated hypersensitive response-associated cell death in Arabidopsis thaliana.

Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae.

BACKGROUND: All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. RESULTS: Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. CONCLUSION: By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

The telomerase inhibitor telomestatin induces telomere shortening and cell death in Arabidopsis.

The cellular response to telomere dysfunction in plants was investigated with the use of telomestatin, an inhibitor of human telomerase activity. Telomestatin bound to plant telomeric repeat sequence, and inhibited telomerase activity in suspension-cultured cells of Arabidopsis thaliana and Oryza sativa (rice) in a dose-dependent manner. The inhibitor did not affect transcript level of the TERT gene, which encodes the catalytic subunit of telomerase, in the plant cells. Inhibition of telomerase activity by telomestatin resulted in rapid shortening of telomeres and the induction of cell death by an apoptosis-like mechanism in Arabidopsis cells. These results suggest that telomerase contributes to the survival of proliferating plant cells by maintaining telomere length, and that telomere erosion triggers cell death.

Enhanced dihydroflavonol-4-reductase activity and NAD homeostasis leading to cell death tolerance in transgenic rice.

The maize Hm1 gene encoding the NADPH-dependent HC-toxin reductase is capable of detoxifying HC-toxin of fungus Cochliobolus carbonum. Here, we conducted the metabolic and biochemical analysis in transgenic rice plants overexpressing an HC-toxin reductase-like gene in rice (YK1 gene). Methods employing NADPH oxidation and capillary electrophoresis mass spectrometry analysis confirmed that YK1 possessed dihydroflavonol-4-reductase activity in vitro and in vivo. The overexpression of YK1 in both suspension-cultured cells and rice plants increased NAD(H) and NADP(H) levels by causing an increase in NAD synthetase and NAD kinase activities. Activity changes in enzymes that require NAD(P) as coenzymes were also noted in rice cells ectopically expressing YK1, where the cell death caused by hydrogen peroxide and bacterial disease was down-regulated. Thus, a strategy was proposed that the combination of dihydroflavonol-4-reductase activity and the elevated level of NAD(P)H pool may confer the prevention of induced cell death in planta.

Characterization of Oryza sativa telomerase reverse transcriptase and possible role of its phosphorylation in the control of telomerase activity.

Telomerase reverse transcriptase (TERT) has been characterized in the dicotyledon Arabidopsis thaliana. A TERT homolog has now been identified in the monocotyledon rice (Oryza sativa L.) on the basis of its predicted homology to the A. thaliana enzyme (AtTERT). At least five alternatively spliced transcripts of the rice TERT (OsTERT) gene were detected. The full-length OsTERT protein shares structural features with TERTs of other species, including a calculated molecular size of 144 kDa, an isoelectric point of 9.6, and conserved sequence motifs. Phylogenetic analysis showed that OsTERT clusters with AtTERT and is more related to the human and mouse enzymes than to those of yeast and ciliated protozoa, consistent with the evolutionary relations among these eukaryotes. Telomerase activity was abundant in shoot apices and cultured cells but was low or absent in leaves or roots of rice plants, whereas similarly spliced OsTERT transcripts were detected in all tissues examined and cultured cells. Similar to mouse and human TERT proteins, OsTERT contains two putative phosphorylation sites for Akt kinase. Incubation of a rice cell extract with Akt or with protein phosphatase 2A potentiated or inhibited telomerase activity, respectively, whereas Akt did not affect the activity in Arabidopsis cell extract. In addition, the kinase activated the telomerase in a leaf extract. The mechanism of telomerase regulation in rice thus appears to differ from that in Arabidopsis (which is mediated predominantly at the level of AtTERT transcription), possibly reflecting the taxonomic distance between monocotyledons and dicotyledons.

Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks.

In higher organisms such as mammals and plants, DNA double-strand breaks (DSBs) are repaired preferentially by non-homologous end joining (NHEJ) rather than by homologous recombination. The NHEJ pathway is mediated by Ku, a heterodimer of approximately 70 and 80 kDa subunits, which contributes to various aspects of the metabolism of DNA ends in eukaryotic cells. On the basis of their predicted sequence similarity to human Ku70 and Ku80, cDNAs encoding the first plant homologues of these proteins (AtKu70 and AtKu80, respectively) have now been isolated from Arabidopsis thaliana. AtKu70 and AtKu80 share 28.6 and 22.5% amino acid sequence identity with human Ku70 and Ku80, respectively. Yeast two-hybrid analysis demonstrated that AtKu70 and AtKu80 form a heterodimer, and electrophoretic mobility-shift assays revealed that this heterodimer binds to double-stranded telomeric and non-telomeric DNA sequences, but not to single-stranded DNA. The AtKu heterodimer also possesses single-stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. Reverse transcription and the polymerase chain reaction revealed that AtKu70 and AtKu80 genes are expressed widely but at low levels in plant tissues. The expression of these two genes in cultured cells was markedly increased in response to the generation of DSBs by bleomycin or methylmethane sulfonate. These results suggest that the evolutionarily conserved Ku70-Ku80 heterodimer functions in DSB repair by the NHEJ pathway in A. thaliana.