天知 誠吾

Intrasporangium sp. strain DVR is an actinobacterium of the family Intrasporangiaceae isolated from soil in Japan. Here we report the draft genome sequence of strain DVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Pelosinus sp. strain IPA-1 is a bacterium isolated from arsenic-contaminated soil in Japan. We here report the draft genome sequence of strain IPA-1.

Microbially influenced corrosion (MIC) may contribute significantly to corrosion-related failures in injection wells and iron pipes of iodine production facilities. In this study, the iron (Fe0) corroding activity of strain Q-1 isolated from iodide-rich brine in Japan and two Iodidimonas strains phylogenetically related to strain Q-1 were investigated under various culture conditions. Under aerobic conditions, the Fe0 foil in the culture of strain Q-1 was oxidized in the presence of nitrate and yeast extract, while those of two Iodidimonas strains were not. The amount of oxidized iron in this culture was six times higher than in the aseptic control. Oxidation of Fe0 in aerobic cultures of nitrate-reducing bacterium Q-1 was dependent on the formation of nitrite from nitrate. This Fe0 corrosion by nitrate-reducing bacterium Q-1 started after initial nitrite accumulation by day 4. Nitrate reduction in strain Q-1 is a unique feature that distinguishes it from two known species of Iodidimonas. Nitrite accumulation was supported by the encoding of genes for nitrate reductase and the missing of genes for nitrite reduction to ammonia or nitrogen gas in its genome sequence. Phylogenetic position of strain Q-1 based on the 16S rRNA gene sequence was with less than 96.1% sequence similarity to two known Iodidimonas species, and digital DNA-DNA hybridization (dDDH) values of 17.2-19.3%, and average nucleotide identity (ANI) values of 73.4-73.7% distinguished strain Q-1 from two known species. In addition of nitrate reduction, the ability to hydrolyze aesculin and gelatin hydrolysis and cellular fatty acid profiles also distinguished strain Q-1 from two known species. Consequently, a new species, named Iodidimonas nitroreducens sp. nov., is proposed for the nitrate-reducing bacterium strain Q-1T.

Azoarcus sp. DN11 was previously isolated from gasoline-contaminated groundwater as an anaerobic benzene-degrading bacterium. Genome analysis of strain DN11 revealed that it contained a putative idr gene cluster (idrABP1P2 ), which was recently found to be involved in bacterial iodate (IO3 -) respiration. In this study, we determined if strain DN11 performed iodate respiration and assessed its potential use to remove and sequester radioactive iodine (129I) from subsurface contaminated aquifers. Strain DN11 coupled acetate oxidation to iodate reduction and grew anaerobically with iodate as the sole electron acceptor. The respiratory iodate reductase (Idr) activity of strain DN11 was visualized on non-denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis of the active band suggested the involvement of IdrA, IdrP1, and IdrP2 in iodate respiration. The transcriptomic analysis also showed that idrA, idrP1 , and idrP2 expression was upregulated under iodate-respiring conditions. After the growth of strain DN11 on iodate, silver-impregnated zeolite was added to the spent medium to remove iodide from the aqueous phase. In the presence of 200 μM iodate as the electron acceptor, more than 98% of iodine was successfully removed from the aqueous phase. These results suggest that strain DN11 is potentially helpful for bioaugmentation of 129I-contaminated subsurface aquifers.

The genus Iodidimonas was recently proposed in the class Alphaproteobacteria. Iodidimonas strains are aerobic, mesophilic, neutrophilic, moderately halophilic, and chemo-organotrophic. They were first discovered in natural gas brine water containing a very high level of iodide (I-). They exhibited a unique phenotypic feature of iodide oxidation to form molecular iodine (I2). Iodidimonas was also enriched and isolated from surface seawater supplemented with iodide, and it is clearer now that their common habitats are those enriched with iodide. In such environments, Iodidimonas species seem to attack microbial competitors with the toxic form I2 to occupy their ecological niche. The iodide-oxidizing enzyme (IOX) purified from the Iodidimonas sp. strain Q-1 exhibited high catalytic efficiency for iodide and consisted of at least two proteins IoxA and IoxC. IoxA is a putative multicopper oxidase with four conserved copper-binding regions but is phylogenetically distinct from other bacterial multicopper oxidases. The IOX/iodide system could be used as a novel enzyme-based antimicrobial system which can efficiently kill Bacillus spores. Furthermore, the IOX/iodide system can be applied to the decolorization of recalcitrant dyes, where iodide may function as a novel inorganic natural redox mediator.

Previously, we found that a multicopper oxidase (IOX) produced by Iodidimonas sp. Q-1, an iodide (I-)-oxidizing marine bacterium, exhibited significant decolorization activity toward various anionic dyes. In this study, the potential capacity of IOX for decolorization of cationic dyes such as malachite green (MG), crystal violet (CV), and methylene blue (MB) was determined. Decolorization of the dyes by IOX exhibited significant pH dependence, and effective decolorization was observed under alkaline conditions. At an optimum pH of 9.5, IOX decolorized more than 90% of MG, CV, and MB within 30 min, 2 h, and 6 h, respectively. The addition of iodide was indispensable for decolorization, suggesting that this halide ion serves as a redox mediator. Decolorization products of MG showed less toxicity than MG against Escherichia coli cells. These results suggest that this IOX-iodide system can be used for the decolorization and detoxification of cationic dyes under alkaline conditions.

AIMS: This study aimed at obtaining a novel fructooligosaccharides (FOS)-producing yeast, which was different from conventional FOS producers, Aureobasidium spp. METHODS AND RESULTS: Strain Him3 was newly isolated from a Japanese dried sweet potato as a FOS producer. The strain exhibited yeast-like cells and melanization on the potato dextrose agar medium, and formed very weak pseudomycelia on the yeast extract polypeptone dextrose agar medium. Based on the internal transcribed spacer (ITS) region of ribosomal DNA and a partial β-tubulin gene sequences, the strain Him3 was identified as Zalaria sp. The β-fructofuranosidase (FFase) produced by strain Him3 was localized on the cell surface (CS-FFase) as well as in the culture broth (EC-FFase). The FOS production yields by CS-FFase and EC-FFase from 50% sucrose were 63.8% and 64.6%, respectively, to consumed sucrose after the reaction for 72 h. CONCLUSIONS: We successfully isolated a novel black yeast, Zalaria sp. Him3, with effective capacity for FOS production. Phylogenetic analysis revealed that strain Him3 was distantly related with the conventional FOS producers, Aureobasidium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Since FFase of strain Him3 demonstrated high production yields of FOS, it could be applied to novel industrial production of FOS, which is different from conventional methods.

A novel dissimilatory antimonate [Sb(V)]-reducing bacterium, strain SVR, was isolated from soil of a former antimony (Sb) mine. Strain SVR coupled Sb(V) reduction to acetate oxidation with an apparent reduction rate of 2.4 mM d-1. The reduction of Sb(V) was followed by the precipitation and accumulation of white microcrystals in the liquid medium. The precipitates were initially small and amorphous, but they eventually developed to the crystal phase with a length > 50 µm. Strain SVR removed 96% of dissolved Sb as the precipitates. An X-ray diffraction analysis indicated that the microcrystals were the orthorhombic Sb trioxide (Sb2O3), i.e., valentinite. Phylogenetic and physiological analyses revealed that strain SVR is a member of the genus Geobacter. The cell suspension of strain SVR incubated with acetate and Sb(V) at pH 7.0 was able to form valentinite. Interestingly, at pH 8.0, the cell suspension formed another crystalline Sb2O3 with a cubic structure, i.e., senarmontite. Our findings provide direct evidence that Geobacter spp. are involved in Sb(V) reduction in nature. Considering its superior capacity for Sb removal, strain SVR could be used for the recovery of Sb and the individual productions of valentinite and senarmontite from Sb-contaminated wastewater.

An anaerobic microbial consortium capable of reductively dehalogenating 2,4,6-triiodophenol (2,4,6-TIP) was enriched from the marine sponge Hymeniacidon sinapium. The enrichment reductively deiodinated 100 μM of 2,4,6-TIP to 4-iodophenol (4-IP) and 2-iodophenol (2-IP) in the presence of sterile sponge tissue as the sole carbon source and electron donor. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis revealed that bacteria closely related with Vallitalea guaymasensis and Oceanirhabdus sediminicola, both of which are members of the order Clostridiales, were predominant in the enrichment. When glucose was added to the enrichment as alternative carbon source, one of these bacteria grew predominantly, which was subsequently isolated as a pure culture. The strain, designated as TIP-1, showed 99.7% 16S rRNA gene sequence similarity with V. guaymasensis. In the presence of glucose, strain TIP-1 reductively deiodinated 2,4,6-TIP to 2-IP and 4-IP at a molar ratio of 3:1, during which 2,4-diiodophenol (2,4-DIP) and 2,6-diiodophenol (2,6-DIP) were observed as deiodinated intermediates. Glucose was required for 2,4,6-TIP deiodination, but 2,4,6-TIP was not essential for growth of strain TIP-1. The strain also deiodinated 2,4-DIP to 2-IP and 4-IP at a molar ratio of 1:1, and 2,6-DIP to 2-IP, but further deiodination of the monoiodophenols was not observed. These results suggest that strain TIP-1 removed both ortho- and para-substituted iodines equally. Such deiodinating bacteria could be applied to the mineralization or dehalogenation of triiodobenzene derivatives, which are widely used as X-ray contrast media.

Chemo-organotrophic iodide (I-)-oxidizing bacterial strains Hi-2T and Mie-1 were isolated from iodide-rich natural gas brine water in Chiba and surface seawater in Mie, Japan, respectively. Cells of strains Hi-2T and Mie-1 were aerobic, Gram-negative and rod-shaped (0.3-0.5 µm width and 1.2-4.4 µm in length). Two isolates grew optimally at 30 °C, pH 7.5 and with 3% NaCl (w/v). Iodide oxidation to form molecular iodine (I2) was a biochemically unique trait for strains Hi-2T and Mie-1. The major cellular fatty acids are C18:1ω7c, C16:1ω5c and C18:1 2-OH. A phylogenetic analysis based on the 16S rRNA gene sequence revealed that strains Hi-2T and Mie-1 were located near Iodidimonas muriae C-3T with 99.2% sequence similarity. The calculated digital DNA-DNA hybridization (dDDH) value of 65.7-65.9% between the two isolates and I. muriae C-3T was lower than the threshold of 70%, which was used for prokaryotic species delineation. Strains Hi-2T and Mie-1 differed in the hydrolysis of aesculin, the hydrolysis of gelatin and the major cellular fatty acids composition from I. muriae C-3T. Considering these biochemical properties, the major cellular fatty acids composition and dDDH value, a novel species is proposed for strains Hi-2T (= JCM 17844T = LMG 28661T) and Mie-1 (= JCM 17845 = LMG 28662), to be named Iodidimonas gelatinilytica.

We report here the complete genome sequence of Geobacter sp. strain SVR, isolated from antimony mine soil in Nakase Mine, Hyogo Prefecture, Japan. SVR strains proliferate using antimonate [Sb(V)] as an electron acceptor, providing insights into the antimony reduction mechanism.

Anaeromyxobacter sp. strain PSR-1, a dissimilatory arsenate [As(V)]-reducing bacterium, can utilize As(V) as a terminal electron acceptor for anaerobic respiration. A previous draft genome analysis revealed that strain PSR-1 lacks typical respiratory As(V) reductase genes (arrAB), which suggested the involvement of another protein in As(V) respiration. Dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions, and was localized predominantly in the periplasmic fraction. The activity was visualized on partially-denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis identified proteins involved in the active band. Among these proteins, a protein annotated as molybdopterin-dependent oxidoreductase (PSR1_00330) exhibited the highest sequence coverage of 76%. Phylogenetic analysis revealed that this protein was a homolog of tetrathionate reductase catalytic subunit TtrA. However, the crude extract of strain PSR-1 did not show significant tetrathionate reductase enzyme activity. Comparative proteomic analysis revealed that the protein PSR1_00330 and a homolog of tetrathionate reductase electron transfer subunit TtrB (PSR1_00329) were expressed abundantly and specifically under As(V)-respiring conditions, respectively. The genes encoding PSR1_00330 and PSR1_00329 formed an operon-like structure along with a c-type cytochrome gene, and their transcription was upregulated under As(V)-respiring conditions. These results suggest that the protein PSR1_00330, which lacks tetrathionate reductase activity, functions as a dissimilatory As(V) reductase in strain PSR-1. Considering the wide distribution of TtrA homologs among bacteria and archaea, they may play a hitherto unknown role along with conventional respiratory As(V) reductase (Arr) in the biogeochemical cycling of arsenic in nature.IMPORTANCE Dissimilatory As(V)-reducing prokaryotes play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. Generally, such prokaryotes reduce As(V) by means of a respiratory As(V) reductase designated as Arr. However, some dissimilatory As(V)-reducing prokaryotes such as Anaeromyxobacter sp. strain PSR-1 lack genes encoding Arr, suggesting the involvement of other protein in As(V) reduction. In this study, by using multiple proteomic and transcriptional analyses, it was found that the dissimilatory As(V) reductase of strain PSR-1 was a protein closely related to the tetrathionate reductase catalytic subunit (TtrA). Tetrathionate reductase is known to play a role in anaerobic respiration of Salmonella on tetrathionate, but strain PSR-1 showed neither growth on tetrathionate nor significant tetrathionate reductase enzyme activity. These results suggest the possibility that TtrA homologs encoded in a wide variety of archaeal and bacterial genomes might function as dissimilatory As(V) reductases.

We report here the draft genome sequence of Geobacter sp. strain SVR, isolated from antimony mine soil in Hyogo Prefecture, Japan. The genome sequence data in this study will provide useful information for understanding bacterial antimonate reduction.

Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of <italic>Anaeromyxobacter</italic> in <italic>Deltaproteobacteria</italic>, commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of <italic>Anaeromyxobacter</italic> in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of <italic>Anaeromyxobacter</italic> to fix nitrogen. Therefore, we verified the diazotrophy of <italic>Anaeromyxobacter</italic> based on both genomic and culture-dependent analyses using <italic>Anaeromyxobacter</italic> sp. PSR-1 and Red267 isolated from soils. Based on the comparison of <italic>nif</italic> gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic <italic>Geobacter</italic> and <italic>Pelobacter</italic> in <italic>Deltaproteobacteria</italic> contain the minimum set of genes for nitrogenase (<italic>nifBHDKEN</italic>). These results imply that <italic>Anaeromyxobacter</italic> species have the ability to fix nitrogen. In fact, <italic>Anaeromyxobacter</italic> PSR-1 and Red267 exhibited N₂-dependent growth and acetylene reduction activity (ARA) in vitro. Transcriptional activity of <italic>nif</italic> gene was also detected when both strains were cultured with N₂ gas as a sole nitrogen source, indicating that <italic>Anaeromyxobacter</italic> can fix and assimilate N₂ gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains based on RNA-based analysis, demonstrating that <italic>Anaeromyxobacter</italic> can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e. nitrogen fixation, of <italic>Anaeromyxobacter</italic>, which is a common soil bacterium. <bold>Importance</bold> <italic>Anaeromyxobacter</italic> is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of <italic>Anaeromyxobacter</italic> from various soil environments. Although the importance of <italic>Anaeromyxobacter</italic> as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genome- and culture-based analyses that <italic>Anaeromyxobacter</italic> fixes nitrogen. This study demonstrates that <italic>Anaeromyxobacter</italic> harboring nitrogenase genes exhibits diazotrophic ability; moreover, N₂-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for <italic>Anaeromyxobacter</italic> to survive under nitrogen-deficient environments, and provide a novel insight into the environmental function of <italic>Anaeromyxobacter</italic>, which is a common bacterium in soils.

Pseudomonas sp. strain SCT is capable of using iodate (IO3- ) as a terminal electron acceptor for anaerobic respiration. A possible key enzyme, periplasmic iodate reductase (Idr), was visualized by active staining on non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that at least four proteins, designated as IdrA, IdrB, IdrP1 , and IdrP2 , were involved in Idr. IdrA and IdrB were homologues of catalytic and electron transfer subunits of respiratory arsenite oxidase (Aio); however, IdrA defined a novel clade within the dimethylsulfoxide (DMSO) reductase family. IdrP1 and IdrP2 were closely related to each other and distantly related to cytochrome c peroxidase. The idr genes (idrABP 1 P 2 ) formed an operon-like structure, and their transcription was upregulated under iodate-respiring conditions. Comparative proteomic analysis also revealed that Idr proteins and high affinity terminal oxidases (Cbb3 and Cyd), various H2 O2 scavengers, and chlorite (ClO2- ) dismutase-like proteins were expressed specifically or abundantly under iodate-respiring conditions. These results suggest that Idr is a respiratory iodate reductase, and that both O2 and H2 O2 are formed as by-products of iodate respiration. We propose an electron transport chain model of strain SCT, in which iodate, H2 O2 , and O2 are used as terminal electron acceptors.

A multicopper oxidase (IOX) produced by Iodidimonas sp. Q-1 has high catalytic efficiency for iodide (I-) oxidation to form molecular iodine (I2). In this study, the potential capacity of IOX for decolorization of recalcitrant dyes was determined. Although IOX did not decolorize any dyes in the absence of redox mediator, significant decolorization of Orange G, Indigo Carmine, Amido Black, and Remazol Brilliant Blue R (RBBR) was observed in the presence of iodide. Addition of 0.1 mM iodide was sufficient to decolorize a total of 3 mM Indigo Carmine, suggesting that iodide functions as a mediator. Such mediator-like function of iodide was not observed in commercially available fungal laccases. The IOX-iodide decolorization system showed much alkaline pH optima of 5.5-6.5 and stronger salt tolerance than fungal laccases did. In addition, actual wastewater discharged from a dyeing factory could be decolorized more than 50% by the system. Since iodide is naturally occurring, non-Toxic, and cheaper than common synthetic mediators, the IOX-iodide system is potentially more advantageous than fungal laccase-mediator systems for decolorization of recalcitrant dyes.

Previously, we hypothesized that microbial laccase oxidizes iodide (I−) in soils to molecular iodine (I2) or hypoiodous acid (HIO), both of which are easily incorporated into natural soil organic matter, and thus plays a role in iodine sorption on soils. In this study, soil iodide oxidase activity was determined by a colorimetric assay to evaluate if laccase is responsible for iodide oxidation in soils. Three types of Japanese soil showed significant iodide oxidase activities (0.751–2.87 mU g soil−1) at pH 4.0, which decreased with increasing pH, until it was no longer detected at pH 5.5. The activity was inhibited strongly by autoclaving or by the addition of common laccase inhibitors. Similar tendency of inhibition was observed in soil laccase activity, which was determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Significant positive correlations (R2 values of 0.855–0.896) between iodide oxidase activity and laccase activity were observed in two of three soils. Commercially available fungal laccases showed only very low iodide oxidase activities (4.68–18.0 mU mg−1), but enhanced activities of 102–739 mU mg−1 were observed in the presence of redox mediators. Finally, we successfully isolated fungal strains with iodide-oxidizing phenotype in the presence of redox mediators. Polyacrylamide gel electrophoresis of the culture supernatant of Scytalidium sp. strain UMS and subsequent active stain revealed that the fungal laccase actually oxidized iodide in the presence of redox mediators. These results suggest that at least part of iodide in soils is oxidized by fungal laccase through the laccase-mediator system. • Iodide oxidase activity in soils showed positive relationships with laccase activity.• Commercial fungal laccases oxidized iodide in the presence of redox mediators.• Iodide-oxidizing fungal strains were newly isolated from soils.• One of strains possessed laccase with iodide oxidase activity.

Microbially mediated arsenate (As(V)) and Fe(III) reduction play important roles in arsenic (As) cycling in nature. Extracellular electron shuttles can impact microbial Fe(III) reduction, yet little is known about their effects on microbial As mobilization in soils. In this study, microcosm experiments consisting of an As-contaminated soil and microbial communities obtained from several pristine soils were conducted, and the effects of electron shuttles on As mobilization were determined. Anthraquinone-2,6-disulfonate (AQDS) and riboflavin (RF) were chosen as common exogenous and biogenic electron shuttles, respectively, and both compounds significantly enhanced reductive dissolution of As and Fe. Accumulation of Fe(II)-bearing minerals was also observed, which may lead to re-immobilization of As after prolonged incubation. Interestingly, Firmicutes-related bacteria became predominant in all microcosms, but their compositions at the lower taxonomic level were different in each microcosm. Putative respiratory As(V) reductase gene (arrA) analysis revealed that bacteria closely related to a Clostridia group, especially those including the genera Desulfitobacterium and Desulfosporosinus, might play significant roles in As mobilization. These results indicate that the natural soil microbial community can use electron shuttles for enhanced mobilization of As; the use of this type of system is potentially advantageous for bioremediation of As-contaminated soils. (C) 2017 Elsevier B.V. All rights reserved.

To identify yeasts involved in white-colony formation on Japanese commercial kimchi products, three types of kimchi were prepared and fermented at four different temperatures. At 4 °C, yeast colonies did not appear until 35 days, while more rapid white-colony formation occurred at higher temperatures (10, 15, and 25 °C). Combination of PCR-DGGE and direct isolation of yeasts from white colonies revealed that Kazachstania exigua and K. pseudohumilis were responsible for the white-colony formation. Inoculation of the isolated Kazachstania strains into fresh kimchi successfully reproduced white-colony formation at 15 °C but not at 4 °C. Growth experiments in liquid medium revealed that Kazachstania spp. grew fast at 15 °C even in the presence of acidulants, which are commonly added to Japanese kimchi products for prevention of yeast growth. These results suggest that white-colony formation on Japanese kimchi is caused by the genus Kazachstania, and that one of important factors determining white-colony formation is its fermentation temperature.

Iodine is a biophilic element that is important for human health, both as an essential component of several thyroid hormones and, on the other hand, as a potential carcinogen in the form of radioiodine generated by anthropogenic nuclear activity. Iodine exists in multiple oxidation states (-1, 0, +1, +3, +5, and +7), primarily as molecular iodine (I-2), iodide (I-), iodate (IO3-), or organic iodine (org-I). The mobility of iodine in the environment is dependent on its speciation and a series of redox, complexation, sorption, precipitation, and microbial reactions. Over the last 15 years, there have been significant advances in iodine biogeochemistry, largely spurred by renewed interest in the fate of radioiodine in the environment. We review the biogeochemistry of iodine, with particular emphasis on the microbial processes responsible for volatilization, accumulation, oxidation, and reduction of iodine, as well as the exciting technological potential of these fascinating microorganisms and enzymes.

A chemo-organotrophic iodide (I-)-oxidizing bacterial strain, C-3T, isolated from natural gas brine of an iodine recovery facility in Kujukuri, Chiba, Japan, was characterized for representation of a novel species in the class Alphaproteobacteria. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the nearest neighbours of strain C-3T were members of the genera Eilatimonas, Kordiimonas, Rhodothalassium and Temperatibacter with 88-91 % sequence similarity. Cells of strain C-3T were aerobic, Gram-staining-negative, non-sporulating and rod-shaped (1.3-3.6 µm in length). Strain C-3T grew optimally at 30 °C, pH 7.5 and with 3 % NaCl (w/v). Iodide oxidation to form molecular iodine (I2) was a unique trait for strain C-3T, whereas the strain did not utilize iodide as a sole electron donor for chemolithoautotrophic growth. The major isoprenoid quinone was Q-10. The major cellular fatty acids were C18 : 1ω7c and C16 : 1ω5c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminolipids. The G+C content of the genomic DNA was 58.5 mol%. Iodide oxidation and the major cellular fatty acids composition distinguished strain C-3T from phylogenetically related bacteria. On the basis of the phenotypic features and the phylogenetic position, a novel genus and species are proposed for strain C-3T (=JCM 17843T=LMG 28660T), to be named Iodidimonas muriae gen. nov., sp. nov. We also propose to place the distinct sublineages of the genera Iodidimonasgen. nov. and Emcibacter in the orders Iodidimonadales ord. nov. and Emcibacterales ord. nov., respectively, because these genera are located far apart from the order Kordiimonadales and form the distinct lineage in the class Alphaproteobacteria.

A bromate (BrO3-)-rechicing bacterium, designated Rhodococcus sp. strain Br-6, was isolated from soil. The strain reduced 250 mu M bromate completely within 4 days under growth conditions transitioning from aerobic to anaerobic conditions, while no reduction was observed under aerobic and anaerobic growth conditions. Bromate was reduced to bromide (Br-) stoichiometrically, and acetate was requited as an electron donor. Interestingly, bromate reduction by strain Br-6 was significantly dependent on both ferric iron and a redox dye 2,6-dichloroindophenol (DCIP). Cell free extract of strain Br-6 showed a dicumarol-sensitive diaphorase activity, which catalyzes the reduction of DCIP in the presence of NADH. Following abiotic experiments showed that the reduced form of DCIP was reoxidized by ferric iron, and that the resulting ferrous iron reduced bromate abiotically. Furthermore, activity staining of the cell free extract revealed that one of diaphorase isoforms possessed a bromate-reducing activity. Our results demonstrate that strain Br-6 utilizes, multiple redox mediators, that is, DCIP and ferric iron, for bromate reduction. Since the apparent rate of bromate reduction by this strain (60 mu M day(-1)) was 3 orders of magnitude higher than that of known bromate-reducing bacteria, it could be applicable to removal of this probable human carcinogen from drinking water.

The impact of arsenite (As[III]) on the bacterial community structure and diversity in soil was determined by incubating soil slurries with 50, 500, and 5,000 mu M As(III). As(III) was oxidized to arsenate (As[V]), and the microbial contribution to As(III) oxidation was 70-100%. PCR-denaturing gradient gel electrophoresis revealed that soil bacterial diversity decreased in the presence of As(III). Bacteria closely related to the family Bacillaceae were predominant in slurry spiked with 5,000 mu M As(III). The population size of culturable As(III)-resistant bacteria was 37-fold higher in this slurry than in unspiked slurry (p &lt; 0.01), indicating that high levels of As(III) stimulate the emergence of As(III)-resistant bacteria. As(III)-resistant bacteria isolated from slurry spiked with 5,000 mu M As(III) were mainly affiliated with the genus Bacillus; however, no strains showed As(III)-oxidizing capacity. An As(III)-oxidizing bacterial community analysis based on As(III) oxidase gene (aioA) sequences demonstrated that diversity was the lowest in slurry spiked with 5,000 mu M As(III). The deduced AioA sequences affiliated with Alphaproteobacteria accounted for 91-93% of all sequences in this slurry, among which those closely related to Bosea spp. were predominant (48-86%). These results suggest that exposure to high levels of As(III) has a significant impact on the composition and diversity of the soil bacterial community, including the As(III)-oxidizing bacterial community. Certain As(III)-oxidizing bacteria with strong As(III) resistance may be enriched under high As(III) levels, while more sensitive As(III) oxidizers are eliminated under these conditions.

Arenibacter sp. strain C-21, isolated from surface marine sediment of Japan, accumulates iodine in the presence of glucose and iodide (I-). We report here the draft genome sequence of this strain to provide insight into the molecular mechanism underlying its iodine-accumulating ability.

Alphaproteobacterium strain Q-1 produces an extracellular multicopper oxidase (IOX), which catalyzes iodide (I-) oxidation to form molecular iodine (I-2). In this study, the antimicrobial activity of the IOX/iodide system was determined. Both Gram-positive and Gram-negative bacteria tested were killed completely within 5 min by 50 mU mL(-1) of IOX and 10 mM iodide. The sporicidal activity of the system was also tested and compared with a common iodophor, povidone-iodine (PVP-I). IOX (300 mU mL(-1)) killed Bacillus cereus, B. subtilis, and Geobacillus stearothermophilus spores with decimal reduction times of 2.58, 7.62, and 40.9 min, respectively. However, 0.1 % PVP-I killed these spores with much longer decimal reduction times of 5.46, 38.0, and 260 min, respectively. To evaluate the more superior sporicidal activity of the IOX system over PVP-I, the amount of free iodine (non-complexed I-2) was determined by an equilibrium dialysis technique. The IOX system included more than 40 mg L-1 of free iodine, while PVP-I included at most 25 mg L-1 free iodine. Our results suggest that the new enzyme-based antimicrobial system is effective against a wide variety of microorganisms and bacterial spores, and that its strong biocidal activity is due to its high free iodine content, which is probably maintained by re-oxidation of iodide released after oxidation of cell components by I-2.

Here, we report a draft genome sequence of Geobacter sp. strain OR-1, an arsenate-respiring bacterium isolated from Japanese paddy soil. It contained two distinct arsenic islands, one including genes for a respiratory arsenate reductase (Arr) as well as for arsenic resistance (arsD-arsA-acr3-arsR-arrA-arrB) and the second containing only genes for arsenic resistance.

Here, we report a draft genome sequence of Anaeromyxobacter sp. strain PSR-1, an arsenate-respiring bacterium isolated from arsenic-contaminated soil. It contained three distinct arsenic resistance gene clusters (ars operons), while no respiratory arsenate reductase gene (arr) was identified.

Roseovarius sp. strain A-2 is an aerobic heterotrophic bacterium with a capacity for oxidizing iodide ion (I-) to form molecular iodine (I-2). In this study, iodide-oxidizing enzyme of strain A-2 was characterized. The enzyme was an extracellular protein, and Cu2+ ion significantly enhanced the enzyme activity in the culture supernatant. When iodide was used as the substrate, the crude enzyme showed Km and V-max values of 4.78 mM and 25.1 U mg(-1), respectively. The enzyme was inhibited by NaN3\, EDTA, KCN, and o-phenanthroline, and also had significant activities toward p-phenylenediamine and hydroquinone. Tandem mass spectrometric analysis of an active band excised from SDS-PAGE gel revealed that at least two proteins are involved in the enzyme. One of these proteins was closely related with IoxA, a multicopper oxidase previously found as a component of iodide-oxidizing enzyme of Alphaproteobacterium strain Q-1. Furthermore, a terrestrial bacterium Rhodanobacter denitrificans 116-2, which possesses an ioxA-like gene in its genome, was found to oxidize iodide. These results suggest that IoxA catalyzes the oxidation of iodide in phylogenetically distinct bacteria.

A chemolithoautotrophic arsenite-oxidizing bacterium, designated strain KGO-5, was isolated from arsenic-contaminated industrial soil. Strain KGO-5 was phylogenetically closely related with Sinorhizobium meliloti with 16S rRNA gene similarity of more than 99%, and oxidized 5 mM arsenite under autotrophic condition within 60 h with a doubling time of 3.0 h. Additions of 0.01-0.1% yeast extract enhanced the growth significantly, and the strain still oxidized arsenite efficiently with much lower doubling times of approximately 1.0 h. Arsenite-oxidizing capacities (11.2-54.1 mu mol h(-1) mg dry cells(-1)) as well as arsenite oxidase (Aio) activities (1.76-10.0 mU mg protein(-1)) were found in the cells grown with arsenite, but neither could be detected in the cells grown without arsenite. Strain KGO-5 possessed putative aioA gene, which is closely related with AioA of Ensifer adhaerens. These results suggest that strain KGO-5 is a facultative chemolithoautotrophic arsenite oxidizer, and its Aio is induced by arsenic.

Arsenic (As) Eontamination of drinking water and soils poses a threat to a large number of people worldwide, especially in Southeast Asia. The predominant forms of As in soils and aquifers are inorganic arsenate [As(V)] and arsenite [As(III)], with the latter being more mobile and toxic. Thus, redox transformations of As are of great importance to predict its fate in the environment, as well as to achieve remediation of As-contaminated water and soils. Although As has been recognized as a toxic element, a wide variety of microorganisms, mainly bacteria, can use it as an electron donor for autotrophic growth or as an electron acceptor for anaerobic respiration. In addition, As detoxification systems in which As is oxidized to the less toxic form or reduced for subsequent excretion are distributed widely in microorganisms. This review describes current development of physiology, biochemistry, and genomics of arsenic-transforming bacteria. Potential application of such bacteria to removal of As from soils and water is also highlighted. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

An anaerobic microbial consortium capable of reductively dehalogenating 2,4,6-triiodophenol (2,4,6-TIP) to 4-iodophenol was enriched from sediments collected from an iodine-producing industry, Chiba, Japan. In the presence of lactate, the enrichment reductively deiodinated 2,4,6-TIP, 2,4-diiodophenol and 2-iodophenol, suggesting preferential removal of ortho-substituted iodines. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis showed that at least 4 bacteria, including Pseudomonas stutzeri, Clostridium sp., Sedimentibacter sp., and unidentified Chloroflexi bacterium, were predominant in the enrichment. Interestingly, the DGGE band corresponding to the Chloroflexi bacterium disappeared when the enrichment was grown in the absence of 2,4,6-triiodophenol. In addition, the DGGE band with a nearly identical gene sequence was detected in another enrichment that consumed 3-amino-2,4,6-triiodobenzoic acid (ATBA). Phylogenetic analysis of 1416 bp of 16S rRNA gene sequence for this putative deiodinating bacterium revealed that it was closely related (93% sequence similarity) with an anaerobic bacterium MO-CFX2 belonging to the class Anaerolineae, which was recently isolated from subseafloor sediments. The sequence similarities with other known reductive dehalogenating bacteria such as Dehalococcoides mccartyi and Dehalobium chlorocoercia were relatively low (78%-80%). Quantitative PCR analysis targeting specific 16S rRNA gene region of the putative deiodinating bacterium showed that the enrichments consuming 2,4,6-TIP or ATBA contained 18 to 1070 times much higher amounts of 16S rRNA gene copies than those in the enrichments that do not consume these iodoaromatic compounds. These results suggest that a novel anaerobic bacterium in the class Anaerolineae is capable of reductively deiodinating 2,4,6-iodobenzene derivatives. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

海洋における有光層ではヨウ素の還元反応が起きることが知られているが、詳細に関しては不明な点も多く存在している。そのため、本研究では実際の海水を培養し、ヨウ素の化学形態変化を観察した。実験の結果、LED光源ではヨウ素酸イオンの減少は確認できたが、ヨウ化物イオンの増加は見られなかった。一方、太陽光で培養した結果ではヨウ素酸イオンの減少とヨウ化物イオンの増加が見られた。このことから、海洋におけるヨウ素の還元反応は中間体を経た二段階反応であることが推測される。

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 contribution of abiotic and biotic processes to the oxidation of arsenite [As(III)] when anaerobic paddy soils were shifted to oxic conditions was investigated. Soil slurries were first incubated under reducing conditions to allow indigenous arsenic (As) to be dissolved into the liquid phase as As(III), and were then switched to oxic incubation conditions with shaking. One day after switching to oxic incubation, As(III) almost disappeared from the liquid phase without any increase of arsenate [As(V)], suggesting that dissolved As(III) was co-precipitated or adsorbed on the soil solid phase. X-ray adsorption near-edge structure (XANES) analysis revealed that the predominant species of As in the solid phase before the oxic incubation was As(III), ranging from 74 to 85% of total As. After 1 d of the oxic incubation, the proportion of As(III) decreased to 46-47%. This oxidation step was an abiotic process and 28-38% of As(III) was oxidized per day on average. However, the abiotic oxidation ceased within 24 h probably due to passivation of reactive sites on the mineral surface. Afterward, a second slow oxidation step (2.5-2.8% per day on average) became predominant. Interestingly, this step was microbiologically mediated, since it did not occur in sterilized soil slurries. Determination of putative arsenite oxidase gene (aioA) sequences suggested that arsenite-oxidizing bacteria are actually present in our soil slurries. Our results suggest that microbial As(III) oxidation accounts for more than 30% of total As(III) oxidation, and thus it is an important process especially after abiotic oxidation ceases.

A novel arsenate-reducing bacterium, designated strain PSR-1, was isolated from arsenic-contaminated soil. Strain PSR-1 was phylogenetically closely related to Anaeromyxobacter dehalogenans 2CP-1(T) with 16S rRNA gene similarity of 99.7% and coupled the oxidation of acetate with the reduction of arsenate. Arsenate reduction was inhibited almost completely by respiratory inhibitors such as dicumarol and 2-heptyl-4-hydroxyquinoline N-oxide. Strain PSR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, oxygen, and fumarate as electron acceptors. Strain PSR-1 catalyzed the release of arsenic from arsenate-adsorbed ferrihydrite. In addition, inoculation of washed cells of strain PSR-1 into sterilized soil successfully reproduced arsenic release. Arsenic K-edge X-ray absorption near-edge structure (XANES) analysis revealed that the proportion of arsenite in the soil solid phase actually increased from 20% to 50% during incubation with washed cells of strain PSR-1. These results suggest that strain PSR-1 is capable of reducing not only dissolved arsenate but also arsenate adsorbed on the soil mineral phase. Arsenate reduction by strain PSR-1 expands the metabolic versatility of Anaeromyxobacter dehalogenans. Considering its distribution throughout diverse soils and anoxic sediments, Anaeromyxobacter dehalogenans may play a role in arsenic release from these environments.

Dissimilatory As(V) (arsenate)-reducing bacteria may play an important role in arsenic release from anoxic sediments in the form of As(III) (arsenite). Although respiratory arsenate reductase genes (arrA) closely related to Geobacter species have been frequently detected in arsenic-rich sediments, it is still unclear whether they directly participate in arsenic release, mainly due to lack of pure cultures capable of arsenate reduction. In this study, we isolated a novel dissimilatory arsenate-reducing bacterium, strain OR-1, from Japanese paddy soil, and found that it was phylogenetically closely related to Geobacter pelophilus. OR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, and fumarate as electron acceptors. OR-1 catalyzed dissolution of arsenic from arsenate-adsorbed ferrihydrite, while Geobacter metallireducens GS-15 did not. Furthermore, inoculation of washed cells of OR-1 into sterilized paddy soil successfully restored arsenic release. Arsenic K-edge X-ray absorption near-edge structure analysis revealed that strain OR-1 reduced arsenate directly on the soil solid phase. Analysis of putative ArrA sequences from paddy soils suggested that Geobacter-related bacteria, including those closely related to OR-1, play an important role in arsenic release from paddy soils. Our results provide direct evidence for arsenic dissolution by Geobacter species and support the hypothesis that Geobacter species play a significant role in reduction and mobilization of arsenic in flooded soils and anoxic sediments.

ヨウ素は人類の必須元素である一方，放射性ヨウ素のリスクに対する関心も高まっている．ヨウ素はさまざまな生物地球化学的プロセスを経て，地球環境中をダイナミックに循環している．近年，ヨウ素循環への環境微生物の関与が明らかになりつつある．ここでは，無機ヨウ素の酸化・還元を行う細菌について概説する．前者はヨウ素を化学兵器として，後者はヨウ素を呼吸の最終電子受容体として利用している．このような特異な細菌が，放射性ヨウ素を含むヨウ素の環境挙動や動態に寄与している可能性がある．

Iodine is one of essential trace elements for humans and animals, and is a constituent of thyroid hormones thyroxine and triiodothyronine. Insufficient iodine in the diet can cause iodine deficiency disorders such as endemic goiter and cretinism. Iodine has one stable isotope, ¹²⁷I, and several radioisotopes, including ¹²⁹I and ¹³¹I. From a radioecological viewpoint, long-lived ¹²⁹I is of great concern because it is one of the most persistent radionuclides released into the environment from nuclear facilities and nuclear weapon testing. Given its long half-life (15.7 million years), ¹²⁹I is expected to behave like a stable isotope over long time periods and it may accumulate in the human thyroid gland. Therefore, it is important to understand geochemistry of iodine for accurate safety assessments of ¹²⁹I. The predominant chemical forms of iodine in the environment are iodate (IO₃^-; oxidation state, +5), iodide (I^-; oxidation state, -1), and organically bound iodine. Recent studies have demonstrated that chemical forms of iodine are influenced or regulated by environmental organisms, especially bacteria. In this review, bacterially catalyzed iodine chemistries, including volatilization, accumulation, oxidation, reduction, sorption, and reductive dehalogenation of iodine, are summarized.

Laccase oxidizes iodide to molecular iodine or hypoiodous acid, both of which are easily incorporated into natural soil organic matter. In this study, iodide sorption and laccase activity in 2 types of Japanese soil were determined under various experimental conditions to evaluate possible involvement of this enzyme in the sorption of iodide. Batch sorption experiment using radioactive iodide tracer (I-125(-)) revealed that the sorption was significantly inhibited by autoclaving (121 degrees C, 40 min), heat treatment (80 and 100 degrees C, 10 min), gamma-irradiation (30 kGy), N-2 gas flushing, and addition of reducing agents and general laccase inhibitors (KCN and NaN3). Interestingly, very similar tendency of inhibition was observed in soil laccase activity, which was determined using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a substrate. The partition coefficient (K-d: mL g(-1)) for iodide and specific activity of laccase in soils (Unit g(-1)) showed significant positive correlation in both soil samples. Addition of a bacterial laccase with an iodide-oxidizing activity to the soils strongly enhanced the sorption of iodide. Furthermore, the enzyme addition partially restored iodide sorption capacity of the autoclaved soil samples. These results suggest that microbial laccase is involved in iodide sorption on soils through the oxidation of iodide.

Alphaproteobacterium strain Q-1 is able to oxidize iodide (I-) to molecular iodine (I-2) by an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme (IOE-II) produced by this strain was excised from a native polyacrylamide gel, eluted, and purified. TOE-It appeared as a single band (51 kDa) and showed significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. However, at least two bands with much higher molecular masses (150 and 230 kDa) were observed with heat treatment (95 degrees C, 3 min). IOE-II was inhibited by NaN3, KCN, EDTA, and a copper chelator, o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence of strain Q-1 revealed that the products of two open reading frames (loth and IoxC), with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected peptides from IoxA and IoxC with high sequence coverage (32 to 40%). IoxA showed homology with the family of multicopper oxidases and included four copper-binding regions that are highly conserved among various multicopper oxidases. These results suggest that IOE-II is a multicopper oxidase and that it may occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are associated.