Removal of Soluble Strontium via Incorporation into Biogenic Carbonate Minerals by Halophilic Bacterium Bacillus sp. Strain TK2d in a Highly Saline Solution.

Radioactive strontium (90Sr) leaked into saline environments, including the ocean, from the Fukushima Daiichi Nuclear Power Plant after a nuclear accident. Since the removal of 90Sr using general adsorbents (e.g., zeolite) is not efficient at high salinity, a suitable alternative immobilization method is necessary. Therefore, we incorporated soluble Sr into biogenic carbonate minerals generated by urease-producing microorganisms from a saline solution. An isolate, Bacillus sp. strain TK2d, from marine sediment removed >99% of Sr after contact for 4 days in a saline solution (1.0 × 10-3 mol liter-1 of Sr, 10% marine broth, and 3% [wt/vol] NaCl). Transmission electron microscopy and energy-dispersive X-ray spectroscopy showed that Sr and Ca accumulated as phosphate minerals inside the cells and adsorbed at the cell surface at 2 days of cultivation, and then carbonate minerals containing Sr and Ca developed outside the cells after 2 days. Energy-dispersive spectroscopy revealed that Sr, but not Mg, was present in the carbonate minerals even after 8 days. X-ray absorption fine-structure analyses showed that a portion of the soluble Sr changed its chemical state to strontianite (SrCO3) in biogenic carbonate minerals. These results indicated that soluble Sr was selectively solidified into biogenic carbonate minerals by the TK2d strain in highly saline environments.IMPORTANCE Radioactive nuclides (134Cs, 137Cs, and 90Sr) leaked into saline environments, including the ocean, from the Fukushima Daiichi Nuclear Power Plant accident. Since the removal of 90Sr using general adsorbents, such as zeolite, is not efficient at high salinity, a suitable alternative immobilization method is necessary. Utilizing the known concept that radioactive 90Sr is incorporated into bones by biomineralization, we got the idea of removing 90Sr via incorporation into biominerals. In this study, we revealed the ability of the isolated ureolytic bacterium to remove Sr under high-salinity conditions and the mechanism of Sr incorporation into biogenic calcium carbonate over a longer duration. These findings indicated the mechanism of the biomineralization by the urease-producing bacterium and the possibility of the biomineralization application for a new purification method for 90Sr in highly saline environments.

A new fungal isolate, Penidiella sp. strain T9, accumulates the rare earth element dysprosium.

With an aim to develop a highly efficient method for the recovery of rare earth elements (REEs) by using microorganisms, we attempted to isolate dysprosium (Dy)-accumulating microorganisms that grow under acidic conditions from environmental samples containing high concentrations of heavy metals. One acidophilic strain, T9, which was isolated from an abandoned mine, decreased the concentration of Dy in medium that contained 100 mg/liter Dy to 53 mg/liter Dy after 3 days of cultivation at pH 2.5. The Dy content in the cell pellet of the T9 strain was 910 µg/mg of dry cells. The T9 strain also accumulated other REEs. Based on the results of 28S-D1/D2 rRNA gene sequencing and morphological characterization, we designated this fungal strain Penidiella sp. T9. Bioaccumulation of Dy was observed on the cell surface of the T9 strain by elemental mapping using scanning electron microscopy-energy dispersive X-ray spectroscopy. Our results indicate that Penidiella sp. T9 has the potential to recover REEs such as Dy from mine drainage and industrial liquid waste under acidic conditions.

Draft Genome Sequence of Stutzerimonas stutzeri NT-I, Which Reduces Selenium Oxyanions into Elemental Selenium and Volatile Selenium Species.

Stutzerimonas stutzeri strain NT-I effectively reduces selenate and selenite into elemental selenium and volatile selenium species. It is thus a promising biological agent for treatment of selenium-contaminated wastewater. We here report the draft genome sequence of this strain.

Molecular cloning and characterization of the srdBCA operon, encoding the respiratory selenate reductase complex, from the selenate-reducing bacterium Bacillus selenatarsenatis SF-1.

Previously, we isolated a selenate- and arsenate-reducing bacterium, designated strain SF-1, from selenium-contaminated sediment and identified it as a novel species, Bacillus selenatarsenatis. B. selenatarsenatis strain SF-1 independently reduces selenate to selenite, arsenate to arsenite, and nitrate to nitrite by anaerobic respiration. To identify the genes involved in selenate reduction, 17 selenate reduction-defective mutant strains were isolated from a mutant library generated by random insertion of transposon Tn916. Tn916 was inserted into the same genome position in eight mutants, and the representative strain SF-1AM4 did not reduce selenate but did reduce nitrate and arsenate to the same extent as the wild-type strain. The disrupted gene was located in an operon composed of three genes designated srdBCA, which were predicted to encode a putative oxidoreductase complex by the BLASTX program. The plasmid vector pGEMsrdBCA, containing the srdBCA operon with its own promoter, conferred the phenotype of selenate reduction in Escherichia coli DH5α, although E. coli strains containing plasmids lacking any one or two of the open reading frames from srdBCA did not exhibit the selenate-reducing phenotype. Domain structure analysis of the deduced amino acid sequence revealed that SrdBCA had typical features of membrane-bound and molybdopterin-containing oxidoreductases. It was therefore proposed that the srdBCA operon encoded a respiratory selenate reductase complex. This is the first report of genes encoding selenate reductase in gram-positive bacteria.

Diversity of salt-tolerant tellurate-reducing bacteria in a marine environment.

Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.

Bioremediation of cadmium contaminated soil using symbiosis between leguminous plant and recombinant rhizobia with the MTL4 and the PCS genes.

Cadmium contamination in rice grains is one of the important issues in Asian countries. We have developed a novel bio-remediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia. We designed two types of recombinant rhizobia, carrying two genes, synthetic tetrameric metallothionein (MTL4) and cDNA encoding phytochelatin synthase from Arabidopsis thaliana (AtPCS). The MTL4 and AtPCS genes were transferred to Mesorhizobium huakuii subsp. rengei B3, which can infect and form nodules on Chinese milk vetch, Astragalus sinicus. The two genes were fused to the nolB or nifH promoter, which generated nodule specific expression of these genes in strain B3. The two recombinant strains, B3(pMPnolBMTL4nifHPCS) and B3::nifHMTL4(pMPnifHPCS), showed 25 and 12-fold increase in Cd concentration, in the free-living cells, respectively. When these recombinant strains established the symbiotic relationship with A. sinicus, the symbionts increased Cd accumulation in nodules by two-fold in hydroponic culture. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. We also applied these recombinant bacteria to rice paddy soil polluted with Cd (1mgkg(-1) dry weight soil). The accumulation of Cd increased not only in nodules but also in the roots of A. sinicus infected by the recombinant rhizobia. The accumulation of Cd in the plant roots infected by B3(pMPnolBMTL4nifHPCS) achieved three-fold than that by the wild-type B3. After two months of cultivation of the symbiont, a maximum of 9% of Cd in paddy soil was removed. Thus, the symbiosis will be useful in phytoremediation for heavy metals.

Construction of an additional metal-binding site in human metallothionein-2.

We have constructed a new metal-binding site in the human metallothionein-2 (hMT-2), using the protein as a scaffold to investigate the structure and function of metal-binding. Potential metal-binding sites were designed within hMT-2 on the basis of structures generated by homology modeling. Amino acid residues D11, C13, C26 and S28 in the beta-domain of hMT-2 (hMT-2beta) were found, by computer search, to form a potential tetrahedral Cys4 metal-binding site. Six mutant proteins were constructed with the following amino acid substitutions: D11C, S28C and D11C/S28C in hMT-2 and the same mutations in hMT-2beta, respectively. These single-mutant and double-mutant proteins bound one gram atom of cadmium or zinc ions per gram molecule of protein more than the corresponding wild-type proteins. The circular dichroism spectra suggested that the structures of the single-mutant proteins that bound Cd or Zn were similar to that of the D11C/S28C double-mutant proteins. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins. The stability with changes in pH of all the mutant proteins was higher than that of the wild-type proteins, and that of the double-mutant D11C/S28C protein was highest. Consequently, it appears that we were able to create novel proteins that bound metal ions at high density and with high affinity.

APeg3, a novel paternally expressed gene 3 antisense RNA transcript specifically expressed in vasopressinergic magnocellular neurons in the rat supraoptic nucleus.

Vasopressin (VP) and oxytocin (OT) play critical roles in the regulation of salt and water balance, lactation, and various behaviors and are expressed at very high levels in specific magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS). In addition to the cell-specific expression of the VP and OT genes in these cells, there are other transcripts that are preferentially expressed in the VP or OT MCNs. One such gene, paternally expressed gene 3 (Peg3), is an imprinted gene expressed exclusively from the paternal allele that encodes a Kruppel-type zinc finger-containing protein involved in maternal behavior and is abundantly expressed in the VP-MCNs. We report here the robust expression in the VP-MCNs of an RNA, which we designate APeg3 that is transcribed in the antisense direction to the 3' untranslated region of the Peg3 gene. The APeg3 mRNA is about 1 kb in size, and the full-length sequence of APeg3, as determined by 5' and 3' RACE, contains an open reading frame that predicts a protein of 93 amino acids and is predominantly expressed in VP-MCNs. Both Peg3 and APeg3 gene expression in the VP-MCNs increase during systemic hyperosmolality in vivo, demonstrating that both of these genes are osmoregulated.

Draft Genome Sequence of Bacillus selenatarsenatis SF-1T, a Promising Agent for Bioremediation of Environments Contaminated with Selenium and Arsenic.

Bacillus selenatarsenatis sp. nov. strain SF-1(T) is a promising agent for bioremediation of environments contaminated with selenium and arsenic. Here, we report the draft genome sequence of this strain.

Identification of cell-specific messenger ribonucleic acids in oxytocinergic and vasopressinergic magnocellular neurons in rat supraoptic nucleus by single-cell differential hybridization.

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system synthesize high levels of the peptides oxytocin (OT) and vasopressin (VP) in separate cells. We used RT-PCR amplification of the RNA from single-cells dissected from supraoptic nuclei of lactating rats to produce cDNAs from identified OT or VP MCNs, which were used to construct OT- and VP-MCN-specific cDNA libraries. These cDNA libraries were then screened using labeled probes from the OT- and VP-cells' amplified cDNAs. Differentially hybridized colonies were isolated and characterized by slot blot hybridization, Southern blot hybridization, DNA sequencing, and in situ hybridization histochemistry. Using this approach, several novel cell-specific mRNAs were identified in the MCNs. One cell-specific clone, phosphofructokinase-C, was isolated from the OT-cell library, and five cell-specific clones were isolated from the VP-cell library. These were identified as paternally expressed gene (Peg)5/neuronatin, metallothionein III, Peg3, synaptotagmin V, and a 3'-phosphoadenosine 5'-phosphosulfate synthase 2-related mRNA. None of these genes would have been predicted to be differentially expressed in OT and VP MCNs, based on our current knowledge; and hence, this single cell differential gene expression approach has begun to further define the MCN phenotypes by identifying selectively expressed molecules in them.

Interactions of arsenic with human metallothionein-2.

Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments. We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous. The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS. The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes.

A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia.

A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp. rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus. The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene. The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd(2+) and Cu(2+), under the low oxygen condition. The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd(2+), but not Cu(2+) in free-living cells. The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd(2+) in the bacterial cells. The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A. sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene. Moreover the MTL4 protein was detected by immunostaining. By infection of the recombinant B3, A. sinicus established symbiosis with the recombinant B3 that was grown in Cd(2+) and Cu(2+)-polluted soils. The symbionts increased Cd(2+) accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu(2+) accumulation was noted.

Molecular analysis of promoter elements from Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a commercially important microorganism that is used in the production of cheeses, cobalamin (vitamin B(12)), and propionic acid. Although a host-vector system in propionibacteria has been developed, there is little information available on the genetic background of the bacteria. To obtain genetic information to facilitate genetic engineering in propionibacteria, we cloned promoter regions from P. freudenreichii using Escherichia coli as a host at the first screening and a promoter-probe vector, pCVE1, which consists of the cholesterol oxidase (choA) gene from Streptomyces sp. as a reporter gene. Finally, nine clones with strong promoter activities in P. freudenreichii were screened by monitoring the choA gene expression and determining if the nucleotide sequences of the cloned DNA fragment were aligned. The initiation sites of these transcripts were determined by primer extension analysis. The putative consensus sequences corresponding to a -35 and -10 hexamer were found to be specific for P. freudenreichii, but not E. coli or other bacteria. Moreover, a new consensus heptamerous sequence between the -35 and -10 regions, termed the -16 region, was also found. It is possible that the putative consensus heptamer is functional and essential to promoter activity in P. freudenreichii. These results should provide new opportunities for controlled gene expression in P. freudenreichii.

Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii.

Since the chemical synthesis of vitamin B12 requires more than 70 steps, the production of vitamin B12 has been achieved by microorganism fermentation with additional brief chemical modifications. In an effort to increase the productivity of vitamin B12, we tried to express 10 genes belonging to the hem, cob and cbi gene families involved in the synthesis of vitamin B12 in Propionibacterium freudenreichii, which is a known producer of vitamin B12. In a recombinant P. freudenreichii clone that harbored the expression vector containing a cobA, cbiLF, or cbiEGH, we obtained an increase in vitamin B12 production of 1.7-, 1.9-, and 1.5-fold higher, respectively, than that in the microorganism without any cloned genes in the expression vector pPK705. The cobU and cobS genes caused a slight increase in the production of vitamin B12. Furthermore, we achieved multigene expression in P. freudenreichii. In a recombinant P. freudenreichii clone that harbored an exogenous gene, hemA, from Rhodobacter sphaeroides and endogenous hemB and cobA genes, we successfully achieved the production of about 1.7 mg/l vitamin B12, 2.2-fold higher than that produced by P. freudenreichii harboring pPK705.

Toward elucidating the full spectrum of mite allergens--state of the art.

Our research has focused on the molecular design of immunotherapeutic vaccines and the advancement of mite-allergy diagnosis. Here, we describe the research history of the major group 1 and group 2 allergens, immunoelectrophoretic analyses covering the complete spectrum of mite allergens, our results on allergens with distinctive characteristics (a conjunctival congestion-eliciting antigen [LM2], an immunotherapeutic antigen [HM2] with high efficacy and without definite adverse reactions, and a potent T-cell stimulatory antigen [HM1] with secretion of IFN-gamma), the full spectrum and immunochemical properties of the major and other important mite allergens (including our newly described allergens: a pan-allergen [tropomyosin, group 10], a potent T-cell stimulatory allergen [M-177, apolipophorin, group 14] and its peptide fragments Mag1 and Mag3, a moderate IgE-binding allergen [gelsolin/villin, group 16], an EF-hand Ca2+-binding allergen [group 17], and a less IgE-binding allergen [heat shock protein 70]), and prospects for the development of immunotherapeutic and diagnostic agents.

Effective selenium volatilization under aerobic conditions and recovery from the aqueous phase by Pseudomonas stutzeri NT-I.

Selenium is an important rare metal and its recovery from waste and wastewater is necessary for its sustainable utilization. Microbial selenium volatilization is suitable for selenium recovery from industrial wastewater because volatile selenium can be recovered in recyclable forms free from other chemicals. We found that Pseudomonas stutzeri NT-I can aerobically transform selenate, selenite, and biogenic elemental selenium into dimethyldiselenide as well as dimethylselenide; these were temporarily accumulated in the aqueous phase and then transferred into the gaseous phase. The rate of selenium volatilization using strain NT-I ranged 6.5-7.6 µmol/L/h in flask experiments and was much higher than the rates reported previously for other microbes. The selenium volatilization rate accelerated to 14 µmol/L/h in a jar fermenter. Furthermore, 82% of the selenium volatilized using strain NT-I was recovered with few impurities within 48 h in a simple gas trap with nitric acid, demonstrating that strain NT-I is a promising biocatalyst for selenium recovery through biovolatilization from the aqueous phase.

Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen.

Lactic acid bacteria (LAB) represent an attractive delivery vehicle for oral allergy vaccine because of their safety as a food microorganism as well as their potent adjuvant activity triggering anti-allergic immune response. Here, we report the generation of recombinant LAB expressing a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB), and their prophylactic effect in vivo. To facilitate heterologous expression, the codon usage in the Cry j 1 gene was optimized for the host LAB strain Lactobacillus plantarum by the recursive PCR-based exhaustive site-directed mutagenesis. Use of the codon-optimized Cry j 1 cDNA and a lactate dehydrogenase gene fusion system led to a successful production of recombinant Cry j 1 in L. plantarum NCL21. We also found that oral vaccination with the Cry j 1-LAB suppressed allergen-specific IgE response and nasal symptoms in a murine model of cedar pollinosis.

Production of 5-aminolevulinic acid by Propionibacterium acidipropionici TISTR442.

Propionibacterium acidipropionici TISTR442 produced the highest amount of 5-aminolevulinic acid (ALA) when cultivated in medium supplemented with glycine at 18g/l. ALA production correlated with ALA synthase activity, whereas ALA dehydratase activity was maintained at a low level. ALA yield reached 405mg/l after prolonged cultivation for 1 month.

Characterization of Pseudomonas stutzeri NT-I capable of removing soluble selenium from the aqueous phase under aerobic conditions.

Pseudomonas stutzeri strain NT-I was isolated from the drainage wastewater of a selenium refinery plant. This bacterium efficiently reduced selenate to elemental selenium without prolonged accumulation of selenite under aerobic conditions. Strain NT-I was able to reduce selenate completely at high concentrations (up to 10 mM) and selenite almost completely (up to 9 mM). In addition, higher concentrations of selenate and selenite were substantially reduced. Activity was observed under the following experimental conditions: 20-50°C, pH 7-9, and 0.05-20 g L(-1) NaCl for selenate reduction, and 20-50°C, pH 6-9, and 0.05-50 g L(-1) NaCl for selenite reduction. Under anaerobic conditions, selenate was reduced more rapidly, whereas selenite was not reduced at all. The high selenate- and selenite-reducing capability at high concentrations suggested that strain NT-I is suitable for the removal of selenium from high-strength industrial wastewater.

Lipoteichoic acids on Lactobacillus plantarum cell surfaces correlate with induction of interleukin-12p40 production.

Heat-killed cells of Lactobacillus plantarum L-137 are potent inducers of IL-12 in vitro as well as in vivo and have been shown to have antiallergic, antitumor, and antiviral effects through this induction, which leads to a Th1 type immune response. To determine why L-137 cells induce much greater IL-12 production than the type strain Lactobacillus plantarum JCM1149, we examined the differences in their CW components. The L-137 CW was found to have a higher alanine content and IL-12p40 induction was significantly greater in comparison with JCM1149 CW, whereas peptidoglycans isolated from both strains did not cause IL-12p40 induction. Because in purified CW preparations from gram-positive bacteria, the presence of LTA, the major proinflammatory structure on these bacteria, has been known to have high alanine content, we investigated the responsiveness of both strains to anti-LTA antibody by flow cytometry. L-137 cells reacted more with anti-LTA antibody than did JCM1149 cells. Furthermore, derivative strains of L-137, cured of a specific plasmid pLTK11 of the 15 endogenous plasmids in wild-type L-137, had poor responsiveness to anti-LTA antibody and showed lower IL-12p40 inducing activity than the wild-type L-137 with pLTK11. Our results suggest that LTA expression on the cell surface causes IL-12p40 induction, and that the above internal plasmid of L-137 influences LTA synthesis and expression on the cell surface.