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

Article pubs.acs.org/est

Effect of Antibiotics on Redox Transformations of Arsenic and Diversity of Arsenite-Oxidizing Bacteria in Sediment Microbial Communities Shigeki Yamamura,*,† Keiji Watanabe,‡ Wataru Suda,§ Shun Tsuboi,† and Mirai Watanabe† †

Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan ‡ Center for Environmental Science in Saitama, 914 Kamitanadare, Kazo, Saitama 347-0115, Japan § Center for Omics and Bioinformatics, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan S Supporting Information *

ABSTRACT: 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 Grampositive 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.

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INTRODUCTION

Antibiotics, which are extensively used in stockbreeding and aquaculture as well as in human medicine, exert potentially devastating effects on microbial communities. Various antibiotics have been detected in sewage and surface water, and numerous studies have elucidated their occurrence, fate, and effects within the past decade.9 The evolution of antibiotic resistance genes in aquatic bacteria poses a global health risk to humans and animals.10−12 The effects of antibiotic disturbance on biogeochemical cycling of elements are largely unknown, although some antibiotics can reportedly affect primary microbial processes such as nitrogen transformation, methanogenesis, and sulfate reduction.13 Previous studies have shown that bacteria can become simultaneously resistant to both antibiotics and metals.14 Coresistance has been shown to be conferred through multiresistance genetic elements, such as plasmids and transposons, containing resistance genes for antibiotics and metals. Crossresistance, however, is derived from the same genetic

Arsenic (As), a highly toxic metalloid, is ubiquitously distributed in nature, but local concentrations depend upon the geochemical characteristics of the environment and on anthropogenic discharges.1 In aquatic environments, As exists mainly as two inorganic forms, arsenate (As(V)) and arsenite (As(III)), of which As(III) is the more mobile and toxic.2 Microbial redox transformations, namely, As(V) reduction and As(III) oxidation, directly affect the mobility of As and play a key role in biogeochemical As cycling.3 Several studies have demonstrated that dissimilatory As(V) reducers act on solidphase As(V) reduction, triggering the release of As(III) into the aqueous phase under anaerobic conditions.4−7 Previously, we have shown that aerobic As(V) reduction (presumably catalyzed by As-resistant bacteria) might be ubiquitous in natural environments.8 In that study, subsequent microbial oxidation of As(III) was also observed, suggesting that active cycling between redox states of inorganic As occurs via microbial activities, rendering As(V) levels unstable in aerobic environments. This implies that the mobility and fate of As in aquatic environments can substantially change when the microflora is disturbed and the As redox cycle is disrupted. © 2013 American Chemical Society

Received: Revised: Accepted: Published: 350

September 6, 2013 December 6, 2013 December 11, 2013 December 11, 2013 dx.doi.org/10.1021/es403971s | Environ. Sci. Technol. 2014, 48, 350−357

Environmental Science & Technology

Article

(EM), lincomycin (LCM), and ampicillin (ABPC) were tested for their effect on microbial As redox transformations. Cultures were incubated aerobically at 30 °C on a reciprocal shaker at 120 rpm. The experiments were performed in triplicate, and portions of the cultures in each flask were periodically collected. Filtered (0.45 μm filter) and nonfiltered samples were frozen for the later analysis of As speciation and quantification and for molecular biological analysis, respectively. DNA Extraction and Amplification of 16S rRNA and Arsenite Oxidase Genes. Total DNA was separately extracted from replicated cultures using the FastDNA SPIN kit for Soil (Q-Biogene, Carlsbad, CA) according to the manufacturer’s instructions. PCR amplification of bacterial 16S rRNA genes from DNA extracts was performed with primers 27F and 1492R.23 Cycling conditions were as follows: initial denaturation for 10 min at 95 °C, then 30 cycles of 1 min at 95 °C, 1 min at 50 °C, and 2 min at 72 °C, with a final extension step at 72 °C for 10 min after the 30th cycle. Reactions were then cooled to 4 °C. Bacterial As(III) oxidase large subunit genes (aioA; formerly referred to as aoxB/aroA/asoA)24 were amplified using primers aoxBM1-2F and aoxBM3-2R.25 The reaction was performed in a 50 μL volume containing 1−10 ng of template DNA, 0.2 μM each primer, 5 μL of 10 × Ex Taq buffer, 4 μL of dNTP mixture, 1 μL of bovine serum albumin (20 mg mL−1), and 0.25 μL of Takara Ex Taq HS (Takara Bio Inc., Shiga, Japan). Cycling conditions were as follows: an initial 5 min denaturation at 94 °C, then 35 cycles of 1 min at 94 °C, 1 min at 52 °C, and 1 min at 72 °C. This was followed by a final 10 min extension at 72 °C after the 35th cycle. Reactions were then cooled to 4 °C. Clone Library Construction and Phylogenetic Analysis. Clone libraries of 16S rRNA and aioA genes were used to investigate the effects of antibiotics on bacterial community structures and the taxonomic distribution of As(III)-oxidizing bacteria in the cultures. Amplified fragments were cloned using the pGEM-T Easy Vector System with DH5α Escherichia coli (Promega, Madison, WI) in accordance with the manufacturer’s instructions. The cloned DNAs were amplified by PCR using T7 and SP6 promoter primers (Promega). The PCR products, which provided the templates for sequencing, were purified using a MinElute 96 UF PCR purification Kit (Qiagen, Hilden, Germany). The primers 341F and 911R were used for 16S rRNA gene sequencing, while primers aoxBM1-2F and aoxBM3-2R assisted the sequencing of aioA genes. Sequencing was undertaken with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA) and an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Chimera check of 16S rRNA gene sequences was performed using the Bellerophon server with parameters set for Huber− Hugenholtz correction and a window size of 200 nucleotides (http://comp-bio.anu.edu.au/bellerophon/bellerophon.pl).26 Each 16S rRNA gene clone was phylogenetically classified using the RDP classifier tool (http://rdp.cme.msu.edu/classifier/ classifier.jsp) release 10.27 For aioA gene phylogeny, the obtained nucleotide sequences were compared with As(III) oxidase sequences from the GenBank database using BlastX. Nucleotide sequences were translated into amino acid sequences and then aligned with reference sequences using ClustalW. Phylogenetic trees were constructed using the neighbor-joining method with the MEGA5 software package.28 Bootstrap resampling analysis (1000 replicates) was carried out to estimate the confidence of

determinant responsible for resistance to antibiotics and metals. There is growing concern that metal contamination can facilitate dissemination of antibiotic resistance genes in natural environments.14−17 Alternatively, antibiotic contamination may exert positive selective pressure for metal-resistant bacteria. Because As-resistant bacteria play an important role in controlling As speciation,3 accumulation of As-resistant bacteria, and the subsequent changes in speciation, are expected to majorly affect As mobility. To our knowledge, however, no previous studies have investigated the effect of antibiotics on As redox transformations in microflora, although a number of plasmids are known to confer resistance to As and antibiotics.18−20 The objectives of this study were to (1) examine the destabilizing influence of antibiotics on inorganic As transformations mediated by aquatic microbial communities and (2) provide insights into susceptible populations within the communities. We used bacterial communities from two aquatic sediments to study the impact of antibiotics on their structure and activity toward inorganic As.

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EXPERIMENTAL SECTION Characterization of Sediments. The sediments used in this study were collected from a small pond at the National Institute for Environmental Studies and from Lake Kasumigaura, a eutrophic lake in Ibaraki Prefecture, Japan. Carbon and nitrogen contents of the sediments were assayed using a NC analyzer (SUMIGRAPH NC-220F, Sumika Chemical Analysis Service, Ltd., Japan). Analysis of other elements (including As) was conducted on sediment samples digested in acidic mixtures (HClO4/HNO3/HF) as described previously.6 Characteristics of the sediments are summarized in Table S1 of the Supporting Information. As(V) Reduction and As(III) Oxidation Experiments. The sediment samples were washed three times with Tris-HCl buffer (pH 7.2). Following centrifugation (8000g, 10 min), the residual sediments were suspended in equal volumes of the same buffer. To elucidate how antibiotics affect microbial redox transformations of As, the experiments were performed in accordance with a previous study that demonstrated the potential microbial As(V) reduction and As(III) oxidation activities in uncontaminated soils.8 In the pond sediment experiments, 1 mL of sediment suspensions was inoculated into 20 mL of basal salt medium supplemented with 10 mM lactate and 1 mM As(V) or As(III) in 50 mL serum bottles. Chloramphenicol (CP) was added to the cultures to give a final antibiotic concentration of 50 mg L−1. To sufficiently inhibit the growth of nonresistant bacteria, the antibiotic concentration was selected by reference to the MICs of reference strains.21,22 For aerobic cultivation, bottles with silicone plugs were used. For anaerobic cultivation, bottles were sealed with butyl rubber stoppers and aluminum crimp seals, and the head space replaced with N2 gas. In the anaerobic experiments supplemented with As(III), 1 mM nitrate was added as an alternative electron acceptor. Cultures were incubated at 30 °C on a reciprocal shaker at 120 rpm. The bottles (duplicated in each treatment) were periodically removed for sampling. In the Lake Kasumigaura sediment experiments, 5 mL of sediment suspensions were inoculated into 500 mL Erlenmeyer flasks containing 200 mL of the above-described medium. In addition to CP, 50 mg L−1 tetracycline (TC), erythromycin 351

dx.doi.org/10.1021/es403971s | Environ. Sci. Technol. 2014, 48, 350−357


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Figure 1. Effect of CP on aerobic As(V) reduction. Medium containing As(V) was inoculated with suspensions of the sediments collected from a small pond and incubated aerobically in the presence or absence of CP. Symbols represent the mean of duplicate experiments, and error bars show the range of data (CP = chloramphenicol).

Figure 2. Effect of CP on aerobic As(III) oxidation. Medium containing As(III) was inoculated with suspensions of the sediments collected from a small pond and incubated aerobically in the presence or absence of CP. Symbols represent the mean of duplicate experiments, and error bars show the range of data (CP = chloramphenicol).

Figure 3. Effect of CP on anaerobic As(V) reduction. Medium containing As(V) was inoculated with suspensions of the sediments collected from a small pond and incubated anaerobically in the presence or absence of CP. Symbols represent the mean of duplicate experiments, and error bars show the range of data (CP = chloramphenicol).

tree topologies. Nucleotide sequences of 16S rRNA and aioA genes were clustered into OTUs at 97% sequence identity with UCLUST.29 OUT-based diversity indices including Chao1, ACE, Shannon’s diversity, Simpson’s diversity, and Pielou’s evenness were calculated using the vegan package in R. Rarefaction curves were constructed by analytic rarefaction software (V.2.0). Principal coordinates analysis (PCoA) was conducted based on weighted UniFrac distance.30 The nucleotide sequences of partial 16S rRNA and aioA genes obtained in this study have been deposited into the DDBJ, EMBL, and GenBank databases under the following accession numbers: AB732386−AB732920, AB730838− AB731107, and AB838711−AB838977. Analytical Procedures. Multi-element analysis of filtered sediment digestions and cultures was carried out by inductively coupled plasma atomic emission spectrometry (ICAP-750, Nippon Jarrell-Ash, Japan), and inductively coupled plasma atomic mass spectrometry (Agilent 7500cx, Agilent Technol-

ogies, CA). As(V) and As(III) concentrations in the cultures were quantified by ion chromatography (ICS-1600 system; Dionex, CA) equipped with IonPack AS23/AG23 columns coupled to a conductivity detector and UV detector set to 210 nm.

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RESULTS Effect of CP on Microbial Arsenic Redox Transformations. The liquid cultures containing the pond sediment were incubated with As(V) and CP under aerobic conditions. As(V) levels declined rapidly, and concomitant increase in As(III) was observed during the first 2 days for all incubations (Figure 1). In the control experiments without CP, subsequent As(V) levels increased significantly, although As(III) tended to decrease. In cultures incubated with As(III), As(V) similarly increased after a 2 day lag (Figure 2). Previous experiments on microflora from various types of forest soils yielded similar results,8 indicating that both microbial As(V) reduction and 352



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established between As(V) reduced and As(III) formed in all incubations, suggesting that most of the As was reversibly transformed between the inorganic states. Bacterial Community Composition. The structure and composition of the Kasumigaura sediment microbial community in the control and CP-amended experiments (Figure 4) was analyzed by preparing a 16S rRNA gene clone library for each 0, 2, and 4 days of incubation. Prior to incubation (day 0), controls at days 2 and 4 and CP-amended cultures at days 2 and 4 yielded 87, 88, 93, 85, and 94 clones, respectively (Figure S1, Supporting Information). In the initial microbial communities (0 days), approximately half of the clones comprised Proteobacteria, decomposed into 16% Betaproteobacteria, 7% Gammaproteobacteria, and 28% Deltaproteobacteria. After 2 days of incubation, Gammaproteobacteria-related clones dominated in both cultures (56% in control and 55% in CPamended cultures). The proportion of Betaproteobacteria remained constant in the presence of CP (14%), although it measurably increased in control cultures (27%). By 4 days of incubation, the compositions of bacterial communities in the control and CP-amended cultures had diverged markedly. In the control culture, Betaproteobacteria accounted for the large majority of the clones (75%). Alphaproteobacteria-related clones were obtained solely from this culture, although they comprised a small minority of the clones analyzed (3%). In contrast, Bacteroidetes dominated in the CP-amended culture (82%), most of which (96%; 74/77) were classified into the genus Chryseobacterium by RDP classifier with an 80% confidence threshold. Although a small proportion of Betaproteobacteria (5%) were present, other proteobacterial clones were not obtained in this culture. Rarefaction analysis showed that the bacterial sequences in CP-amended culture exhibited relatively lower diversity than control culture at 4 days, although the sequencing was under-saturated in all clone libraries (Figure S2, Supporting Information). Diversity of Arsenite Oxidase Genes. To detect the presence of aioA genes in the Kasumigaura sediment cultures (Figure 4), we used the aoxBM1-2F/3-2R primers on 0, 2, and 4 day cultures. Specific amplifications of about 1100 bp were obtained from the DNA samples of all cultures, except for the 2 day culture which was amended with CP (Figure S3, Supporting Information). The aioA gene clone libraries were constructed from the amplified fragments, and 42−85 aioA-like sequences were obtained from each sample, except for the 4 day culture amended with CP (which yielded just seven sequences). Therefore, the CP-amended culture was excluded from the rarefaction analysis of the clone libraries (Figure 5). Although the clone libraries were not exhaustive, the sequence populations of antibiotics-amended cultures were less diverse than that of the control culture. This observation was supported by the species richness estimators and diversity parameters (Table S2, Supporting Information). Moreover, UniFrac PCoA of the clone libraries showed differential distribution patterns depending on the antibiotics added (Figure 6). The deduced protein products were phylogenetically analyzed against AioA (AoxB/AroA/AsoA) reference sequences. Almost all of the AioA sequences obtained in this study were assigned to two major groups mainly composed of Proteobacteria sequences (Figure S4, Supporting Information, and Figure 7). In the 0 day sample, although a third of the sequences belonged to group I (consisting primarily of Alphaproteobacteria), Beta/Gammaproteobacteria-related group

As(III) oxidation actively occurred in the control experiments. In contrast, CP almost completely inhibited As(III) oxidation in both As(V)- and As(III)-containing cultures (Figures 1 and 2) but exerted no appreciable effect on As(V) reduction (Figure 1). Although the total dissolved As gradually decreased, the sum of As(V) and As(III) approximately equaled the total As in all incubations. Under anaerobic conditions, As(V) was immediately reduced in the control experiments (Figure 3). In contrast to aerobic experiments, CP largely inhibited As(V) reduction. As(III) oxidation was absent in all anaerobic incubations, including cultures supplemented with As(III) (data not shown). Effect of Diverse Antibiotics on Aerobic Arsenic Redox Transformations. The sediments collected from Lake Kasumigaura were incubated in liquid media containing As(V) and different types of antibiotics under aerobic conditions. As observed in the preliminary pond sediment experiments (Figure 1), immediate As(V) reduction and subsequent As(III) oxidation occurred in the control experiments (Figure 4). As(V) reduction was also observed in the

Figure 4. Effect of various antibiotics on aerobic As redox transformations. Medium containing As(V) and antibiotic was inoculated with suspensions of the sediments collected from Lake Kasumigaura and incubated aerobically. Symbols represent the mean of triplicate experiments, and error bars show the standard deviations (CP = chloramphenicol, TC = tetracycline, EM = erythromycin, LCM = lincomycin, and ABPC = ampicillin).

presence of all antibiotics tested. The rate of As(V) reduction was slightly delayed in CP and accelerated in TC cultures. Subsequent As(III) oxidation was inhibited in the presence of CP and delayed in the presence of TC, although activity resumed in the later phase of the TC experiments. The As(V) reduction was incomplete in the ABPC-amended culture, and the effect of ABPC on As(III) oxidation was indeterminable. By contrast, EM and LCM seemed to exert a minor effect on both As(V) reduction and As(III) oxidation. An equivalence was 353



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Figure 5. Rarefaction curves obtained for the aioA gene sequences in clone libraries of 0, 2, and 4 day cultures of the control and antibiotics-amended experiments (N = control, TC = tetracycline, EM = erythromycin, LCM = lincomycin, and ABPC = ampicillin). The vertical axis indicates the number of OTUs clustered at 97% similarity. For experimental conditions, see Figure 4.

microbial As(V) reduction and As(III) oxidation are ubiquitous natural processes. In our experiments using sediment cultures, As(V) reduction was activated under both aerobic and anaerobic conditions. Nonetheless, responses of aerobic and anaerobic As(V) reducers to antibiotics differed markedly. Specifically, the inhibitory effect of CP on As(V) reduction was reduced under aerobic conditions. Dowdle et al. also reported that anaerobic As(V) reduction in saltmarsh sediment incubated with lactate was inhibited by CP.31 Two different bacterial As(V) reduction mechanisms have been reported.3,4,32 Under anaerobic conditions, dissimilatory As(V)-reducing bacteria utilize As(V) as a terminal electron acceptor via a periplasmic or membrane-associated As(V) reductase (Arr).3,32 Alternatively, As(V) reduction as a means of resistance yields no energy to the organism, which can occur under both aerobic and anaerobic conditions.3 The best-characterized As resistance mechanism is encoded by the ars operon, comprising cytoplasmic reductase (ArsC) and subsequent As(III) efflux systems, which is widespread in bacterial plasmids/chromosomes.33 The plasmids containing ars genes frequently confer resistance to CP.19,20,34 Thus, our results imply that As resistance systems such as ars predominate in aerobic As(V) reduction, although the dissimilatory process may play a major role in anaerobic As(V) reduction. It is also possible that aerobic and anaerobic As(V) reduction are catalyzed by constitutive and inducible enzymes, respectively, because CP does not kill bacteria but inhibits their growth by preventing protein synthesis. Addition of CP into aerobic cultures containing Kasumigaura sediment resulted in dominance by Chryseobacterium, a genus that exhibits resistance to a wide range of antibiotics including CP.35,36 ars genes have also been found in some Chryseobacterium strains.37,38 Moreover, aerobic As(V) reduction proceeded even in the presence of diverse types of antibiotics, indicating that As-resistant bacteria are broadly resistant to antibiotics and that bacterial coresistance to As and antibiotics is distributed throughout aquatic environments. In contrast, aerobic As(III) oxidation was more susceptible to certain types of antibiotics. CP in particular inhibited As oxidation in both sediment cultures. Likewise, aioA-related genes were absent or conspicuously few in samples obtained from CP-amended cultures, although bacterial aioA amplification may have been inhibited by the choice of primers used in this study. Although phylogenetically diverse As(III)-oxidizing bacteria exist in numerous environments, most proteobacterial

Figure 6. PCoA of aioA gene sequences in clone libraries generated from 0, 2, and 4 day cultures of the control and antibiotics-amended experiments (N = control, TC = tetracycline, EM = erythromycin, LCM = lincomycin, and ABPC = ampicillin) based on weighted UniFrac distance. For experimental conditions, see Figure 4.

II sequences dominated (Figure S4, Supporting Information). The majority of sequences in both groups were clustered in a specific branch distinct from the reference sequences. After 2 days of incubation, the composition of the deduced AioA sequences in each culture clearly depended on antibiotic exposure (Figure 7). In the control cultures without antibiotics, a number of sequences belonged to group II and the proportion of group I sequences was decreased, although they were distributed among diverse subgroups (II-A, II-B, and II-C). In contrast, the sequences obtained from antibioticsamended cultures were more closely associated with other sequences from the same culture. For example, in the TCamended culture, the majority of sequences clustered in a specific branch associated with Burkholderia multivorans (82− 96% identities). Apart from the LCM-cultured sequences, most sequences were clustered into Gammaproteobacteria-related subgroup (II-A) in group II (Figure 8). After 4 days of incubation, the number of group I-related sequences increased, especially in the control culture, although group II sequences continued to dominate in antibiotics-amended samples.

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DISCUSSION Our previous study has revealed the microbial As(V)-reducing and As(III)-oxidizing activities of various As-free soils.8 In this study, we obtained similar results from aquatic sediments containing background levels of As, indicating that both 354



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Figure 7. Neighbor-joining phylogenetic trees of bacterial AioA protein sequences retrieved from the control (no antibiotics; N) and antibioticsamended cultures. For experimental conditions, see Figure 4. The sequences obtained in this study are shown in underlined bold font. Numbers in parentheses indicate the number of closely related sequences obtained from the same culture. Sequences obtained from antibiotics-amended cultures are colored (purple, CP = chloramphenicol; orange, TC = tetracycline; blue, EM = erythromycin; red, LCM = lincomycin; and green, ABPC = ampicillin). Circles and triangles at the branch nodes represent bootstrap percentages: filled circles, 90−100%; open circles, 70−90%; and open triangles, 50−70%. Values less than 50% are not shown. The scale bar represents the estimated number of substitutions per site. Accession numbers for reference sequences are presented in Figure S4 of the Supporting Information. 355



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Figure 8. Numbers of sequences clustered in the different AioA groups shown in Figure 7.

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AioA-related sequences have been isolated from mesophilic environments.25,39−42 Consistent with these reports, the proportion of Proteobacteria-related 16S rRNA genes obtained in our current study were decreased drastically in CP-amended cultures, whereas they dominated in As(III) oxidizing control cultures. These findings indicate that the large majority of As(III) oxidizers in our experiments are Proteobacteria. The minimal effects of EM and LCM, compared with the other antibiotics tested, on As(III) oxidation further support this hypothesis because these antibiotics primarily act against Grampositive bacteria.13 However, the AioA sequences obtained from EM- and LCM-amended cultures were less diverse than those of control cultures, although almost all of the sequences in the cultures belonged to two major Proteobacteria-related groups, as previously described.25,43 Interestingly, the AioA sequence composition and distribution patterns differed significantly between EM and LCM cultures; nonetheless, the time courses of As(III) oxidation were similar in both cultures. Sequences derived from EM cultures tended to be closely related to Gammaproteobacteria-related subgroup (II-A), whereas the largest number of LCM sequences (72% in 2 day cultures and 59% in 4 day cultures) belonged to a Betaproteobacteriarelated subgroup (II-B). The presence of other antibiotics, e.g., TC and ABPC, induced specific clustering of AioA sequences. The diversity of aioA genes in bacteria inhabiting Ascontaminated environments is now known to be wider than previously suspected.43,44 Moreover, in a recent study, aioA-like genes were also obtained from an uncontaminated soil.45 Our results further indicate that diverse As(III)-oxidizing bacteria reside in the environment regardless of As contamination. Antibiotic response assay is a potentially useful tool for the enrichment and isolation of particular groups of As(III)oxidizing bacteria and As(III) oxidase genes. The results of this study show that susceptibility to antibiotics differs substantially between As(V) reduction and As(III) oxidation in sediment microbial communities and that inactivation of Proteobacteria may accelerate aerobic As(V) reduction via preferential inhibition of As(III) oxidation. This implies that introducing certain type of antibiotics (such as CP) into aquatic environments promotes As mobilization under aerobic conditions, although further detailed studies more closely simulating environmental conditions are required to determine the overall effects of antibiotics on the biogeochemical cycle of As.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-29-850-2168. Fax: +81-29-850-2569. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant 23681005. The authors thank Dr. M. Nishikawa and Ms. Masayo Okawa for their assistance with inductively coupled plasma atomic emission spectrometry analyses, Ms. Megumi Okawa for ion chromatography analyses and clone libraries construction, and Dr. K. Komatsu for collection of the sediment from Lake Kasumigaura. We thank the editor and anonymous reviewers for improving the quality of this manuscript.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1−S2 and Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org. 356



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