Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

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.

A novel dimethylsulfoxide reductase family of molybdenum enzyme, Idr, is involved in iodate respiration by Pseudomonas sp. SCT.

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.

Possible Involvement of a Tetrathionate Reductase Homolog in Dissimilatory Arsenate Reduction by Anaeromyxobacter sp. Strain PSR-1.

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 by 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, 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 gene encoding a c-type cytochrome (cyt c), 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 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, 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.

Soil Microbial Communities Involved in Reductive Dissolution of Arsenic from Arsenate-Laden Minerals with Different Carbon Sources.

The natural microbial communities involved in arsenic (As) extraction under biostimulated conditions are still unclear. In this study, soil slurry was incubated with arsenate [As(V)]-laden Fe(III) or Al (hydr)oxides with lactate or acetate. After 40 d, dissolved As released from As(V)-laden Fe(III) accounted for 54% of the initial solid-phase As in lactate-amended slurries, while much less As was released from acetate-amended slurries. As was released more rapidly from As(V)-laden Al, but the total release was relatively low (45%). High-throughput Illumina sequencing of 16S rRNA genes revealed that dissimilatory metal(loid) reducers such as Desulfitobacterium became predominant in lactate-amended slurries. Moreover, anaerobic fermenters in the Sporomusaceae family were predominant. Interestingly, a Sporomusaceae bacterial strain isolated from the slurry was capable of releasing As from both As(V)-laden (hydr)oxides in the presence of lactate. The strain first released As as As(V) and subsequently reduced it to As(III) in the aqueous phase. These results suggest that lactate is a suitable carbon source for As extraction by natural microbial communities, and that both dissimilatory metal(loid) reducers and certain anaerobic fermenters play significant roles in As extraction. Microbial reductive dissolution of As may be expected to be a cost-effective restoration technique for As-contaminated soils.

Effect of antibiotics on redox transformations of arsenic and diversity of arsenite-oxidizing bacteria in sediment microbial communities.

In the present study, we investigated the effect of antibiotics on microbial arsenate (As(V)) reduction and arsenite (As(III)) oxidation in sediments collected from a small pond and eutrophic lake. The As(V)-reducing activities were less susceptible to chloramphenicol in aerobic conditions than in anaerobic conditions. Aerobic As(V) reduction proceeded in the presence of diverse types of antibiotics, suggesting that As-resistant bacteria are widely antibiotic resistant. In contrast, some antibiotics, e.g., chloramphenicol, strongly inhibited aerobic As(III) oxidation. In addition, bacterial As(III) oxidase genes were scarcely amplified and Proteobacteria -related 16S rRNA genes drastically decreased in chloramphenicol-amended cultures. Erythromycin and lincomycin, which successfully target many Gram-positive bacteria, scarcely affected As(III) oxidation, although they decreased the diversity of As(III) oxidase genes. These results indicate that the aerobic As(III) oxidizers in the sediment cultures are mainly composed of Proteobacteria and are more sensitive to certain types of antibiotics than the aerobic As(V) reducers. Our results suggest that antibiotic disturbance of environmental microbial communities may affect the biogeochemical cycle of As.

Draft genome sequence of Intrasporangium sp. DVR, an actinobacterium of the family Intrasporangiaceae isolated from soil in Japan.

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.

Draft genome sequence of Pelosinus sp. IPA-1, a bacterium isolated from arsenic-contaminated soil in Japan.

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.

Ecological niche separation in the Polynucleobacter subclusters linked to quality of dissolved organic matter: a demonstration using a high sensitivity cultivation-based approach.

The free-living, cosmopolitan, freshwater betaproteobacterial bacterioplankton genus Polynucleobacter was detected in different years in 11 lakes of varying types and a river using the size-exclusion assay method (SEAM). Of the 350 strains isolated, 228 (65.1%) were affiliated with the Polynucleobacter subclusters PnecC (30.0%) and PnecD (35.1%). Significant positive correlations between fluorescence in situ hybridization and SEAM data were observed in the relative abundance of PnecC and PnecD bacteria to Polynucleobacter communities (PnecC + PnecD). Isolates were mainly PnecC bacteria in the samples with a high specific UV absorbance at 254 nm (SUVA(254) ), and a low total hydrolysable neutral carbohydrate and amino acid (THneutralCH + THAA) content of the dissolved organic matter (DOM) fraction, which is known to be correlated with a high humic content. In contrast, the PnecD bacteria were abundant in samples with high chlorophyll a and/or THneutralCH + THAA concentrations, indicative of primary productivity. With few exceptions, differences in the relative abundance of PnecC and PnecD in each sample, determined using a high-sensitivity cultivation-based approach, were due to DOM quality. These results suggest that the major DOM component in the field, which is allochthonously or autochthonously derived, is a key factor for ecological niche separation between PnecC and PnecD subclusters.

Draft Genome Sequence of the Anaerobic Arsenite-Oxidizing Halomonas sp. Strain ANAO-440, Isolated from an Alkaline Saline Lake in Khovsgol, Mongolia.

The draft genome sequence of Halomonas sp. strain ANAO-440 contains 3,866 predicted protein-coding sequences. This strain is capable of anaerobic arsenite oxidation and encodes an arxA-type arsenite oxidase within the arxB2AB1CD gene island. This genome sequence provides valuable information regarding the physiological diversity of Arx-dependent arsenite-oxidizing microorganisms.

Draft Genome Sequence of Cupriavidus sp. Strain IK-TO18, Isolated from Antimony-Contaminated Sediment.

Cupriavidus sp. strain IK-TO18 was isolated from antimony-contaminated sediment. The draft genome sequence of the isolate contains 6,605 predicted protein-coding sequences, including genes associated with heavy metal resistance and the aerobic degradation of aromatic hydrocarbons. This sequence will provide valuable information regarding the functional versatility of the genus Cupriavidus.

Production of two morphologically different antimony trioxides by a novel antimonate-reducing bacterium, Geobacter sp. SVR.

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.

Complete Genome Sequence of Geobacter sp. Strain SVR, an Antimonate-Reducing Bacterium Isolated from Antimony-Rich Mine Soil.

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.

Draft Genome Sequence of the Antimony-Oxidizing Pseudomonas sp. Strain SbOxS1, Isolated from Stibnite Mine Tailing Soil.

Pseudomonas sp. strain SbOxS1 was isolated from stibnite mine tailing soil for its ability to oxidize antimonite. We present a draft genome sequence of strain SbOxS1, which contains 6,484 predicted protein-coding sequences. This genome information extends our understanding of the physiological versatility of antimony-transforming microorganisms.

Draft Genome Sequence of Stenotrophomonas sp. Strain SbOxS2, an Antimony-Oxidizing Bacterium Isolated from Stibnite Mine Tailing Soil.

The antimony-oxidizing Stenotrophomonas sp. strain SbOxS2 was isolated from stibnite mine tailing soil. The draft genome sequence of strain SbOxS2 comprises 4.76 Mbp with 4,211 predicted protein-coding sequences. This genome will provide useful information for characterizing the molecular mechanisms associated with heavy metal resistance within the genus Stenotrophomonas.

Draft Genome Sequence of Geobacter sp. Strain SVR, Isolated from Antimony Mine Soil.

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.

Vertical variation of bulk and metabolically active prokaryotic community in sediment of a hypereutrophic freshwater lake.

This study was conducted to acquire novel insight into differences between bulk (16S rDNA) and metabolically active (16S rRNA) prokaryotic communities in the sediment of a hypereutrophic lake (Japan). In the bulk communities, the class Deltaproteobacteria and the order Methanomicrobiales were dominant among bacteria and methanogens. In the metabolically active communities, the class Alphaproteobacteria and the order Methanomicrobiales and the family Methanosaetaceae were frequently found among bacteria and methanogens. Unlike the bulk communities of prokaryotes, the composition of the metabolically active communities varied remarkably vertically, and their diversities greatly decreased in the lower 20 cm of sediment. The metabolically active prokaryotic community in the sediment core was divided into three sections based on their similarity: 0-6 cm (section 1), 9-18 cm (section 2), and 21-42 cm (section 3). This sectional distribution was consistent with the vertical pattern of the sedimentary stable carbon and nitrogen isotope ratios and oxidation-reduction potential in the porewater. These results suggest that vertical disturbance of the sediment may influence the communities and functions of metabolically active prokaryotes in freshwater lake sediments. Overall, our results indicate that rRNA analysis may be more effective than rDNA analysis for evaluation of relationships between actual microbial processes and material cycling in lake sediments.

Draft Genome Sequence of a Novel Lactate-Fermenting Bacterial Strain of the Family Sporomusaceae within the Class Negativicutes.

Here, we report a draft genome sequence of the Sporomusaceae bacterial strain FL31, a novel lactate-fermenting bacterium of the family Sporomusaceae within the class Negativicutes. This genome furthers our understanding of the physiological functions of this taxonomic group in natural environments.

Genomic Analysis of Pseudomonas sp. Strain SCT, an Iodate-Reducing Bacterium Isolated from Marine Sediment, Reveals a Possible Use for Bioremediation.

Strain SCT is an iodate-reducing bacterium isolated from marine sediment in Kanagawa Prefecture, Japan. In this study, we determined the draft genome sequence of strain SCT and compared it to complete genome sequences of other closely related bacteria, including Pseudomonas stutzeri A phylogeny inferred from concatenation of core genes revealed that strain SCT was closely related to marine isolates of P. stutzeri Genes present in the SCT genome but absent from the other analyzed P. stutzeri genomes comprised clusters corresponding to putative prophage regions and possible operons. They included pil genes, which encode type IV pili for natural transformation; the mer operon, which encodes resistance systems for mercury; and the pst operon, which encodes a Pi-specific transport system for phosphate uptake. We found that strain SCT had more prophage-like genes than the other P. stutzeri strains and that the majority (70%) of them were SCT strain-specific. These genes, encoded on distinct prophage regions, may have been acquired after branching from a common ancestor following independent phage transfer events. Thus, the genome sequence of Pseudomonas sp. strain SCT can provide detailed insights into its metabolic potential and the evolution of genetic elements associated with its unique phenotype.

137Cs transfer from canopies onto forest floors at Mount Tsukuba in the four years following the Fukushima nuclear accident.

This study investigated the transport of 137Cs within a forest ecosystem by examining temporal changes in the inventory and determining the major pathways of transfer following significant atmospheric deposition. A forested area of eastern Japan was monitored for four years immediately after the Fukushima nuclear power plant accident in March 2011 that released a large amount of radionuclides. The long physical half-life of 137Cs means that contamination can persist for decades, so it is vital to understand the mechanisms underlying the 137Cs dynamics in ecosystems. We sampled litterfall, throughfall, and soil, mainly from a cedar stand, over a four-year period, and analyzed the 137Cs concentrations of each sample to determine the transfer rate and total inventory. After validating our methodology through a comparison with results from an earlier study, we determined the temporal changes in the 137Cs distribution and in the major transfer pathway. Results showed that most 137Cs intercepted by canopies was transferred rapidly over the first nine months, and that the major pathway was not litterfall but throughfall. The ecological half-life of the 137Cs stocked in the canopy was calculated for both the early and later stages of contamination. Although the former is consistent with previous results, the latter ecological half-life is somewhat longer, probably because of dependence on the meteorological and tree physiological conditions at the site. This study presents valuable new data on the post-Fukushima 137Cs contamination, enhancing our understanding of the associated dynamics in forest ecosystems.

Genomic Analysis of Rhodococcus sp. Br-6, a Bromate Reducing Bacterium Isolated From Soil in Chiba, Japan.

Bromate is a byproduct of the ozone disinfection of drinking water. It is a genotoxic carcinogen and causes renal cell tumors in rats. Physicochemical removal of bromate is very difficult, making microbial reduction of bromate to bromide a promising approach to eliminate bromate from water. Rhodococcus sp. Br-6, isolated from soil, can efficiently reduce bromate by using acetate as an electron donor. We determined the draft genome sequence of Rhodococcus sp. Br-6 for the potential practical application of the bromate-reducing bacterium. Core genome phylogeny suggests that the Br-6 strain is most closely related to R. equi. The Br-6 genome contains genes encoding multiple isoforms of diaphorase, previously found to be involved in Br-6-mediated bromate reduction. The genes identified in the present study could be effective targets for experimental studies of microbial bromate reduction in the future.