aGraduate School of Horticulture, Chiba University, Matsudo-city, Chiba, JapanbInstitute of Low Temperature Science, Hokkaido University, Sapporo, JapancDepartment of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan
ABSTRACT The reduction of arsenate [As(V)] to arsenite [As(III)] by dissimilatoryAs(V)-reducing bacteria, such as Geobacter spp., may play a significant role in arsenicrelease from anaerobic sediments into groundwater. The biochemical and molecularmechanisms by which these bacteria cope with this toxic element remain unclear. Inthis study, the expression of several genes involved in arsenic respiration (arr) andresistance (ars) was determined using Geobacter sp. strain OR-1, the only culturedGeobacter strain capable of As(V) respiration. In addition, proteins expressed differen-tially under As(V)-respiring conditions were identified by semiquantitative proteomicanalysis. Dissimilatory As(V) reductase (Arr) of strain OR-1 was localized predomi-nantly in the periplasmic space, and the transcription of its gene (arrA) was upregulatedunder As(V)-respiring conditions. The transcription of the detoxifying As(V) reductasegene (arsC) was also upregulated, but its induction required 500 times higher concentra-tion of As(III) (500 �M) than did the arrA gene. Comparative proteomic analysis revealedthat in addition to the Arr and Ars proteins, proteins involved in the following processeswere upregulated under As(V)-respiring conditions: (i) protein folding and assembly forrescue of proteins with oxidative damage, (ii) DNA replication and repair for restorationof DNA breaks, (iii) anaplerosis and gluconeogenesis for sustainable energy productionand biomass formation, and (iv) protein and nucleotide synthesis for the replacement ofdamaged proteins and nucleotides. These results suggest that strain OR-1 copes with ar-senic stress by orchestrating pleiotropic processes that enable this bacterium to resistand actively metabolize arsenic.
IMPORTANCE Dissimilatory As(V)-reducing bacteria, such as Geobacter spp., play sig-nificant roles in arsenic release and contamination in groundwater and threaten thehealth of people worldwide. However, the biochemical and molecular mechanismsby which these bacteria cope with arsenic toxicity remain unclear. In this study, itwas found that both respiratory and detoxifying As(V) reductases of a dissimilatoryAs(V)-reducing bacterium, Geobacter sp. strain OR-1, were upregulated under As(V)-respiring conditions. In addition, various proteins expressed specifically or moreabundantly in strain OR-1 under arsenic stress were identified. Strain OR-1 actively me-tabolizes arsenic while orchestrating various metabolic processes that repair oxidativedamage caused by arsenic. Such information is useful in assessing and identifying possi-ble countermeasures for the prevention of microbial arsenic release in nature.
KEYWORDS Geobacter, arrA, arsC, arsenate reduction, arsenic, proteomics, qRT-PCR
Arsenic is released from both natural and anthropogenic sources and is widelydistributed in the environment. In sediments and groundwater, arsenic can exist in
multiple oxidation states, with the predominant forms being inorganic arsenate [As(V)]
Citation Tsuchiya T, Ehara A, Kasahara Y,Hamamura N, Amachi S. 2019. Expression ofgenes and proteins involved in arsenicrespiration and resistance in dissimilatoryarsenate-reducing Geobacter sp. strain OR-1.Appl Environ Microbiol 85:e00763-19. https://doi.org/10.1128/AEM.00763-19.
and arsenite [As(III)] (1, 2). As(V) exists in ionic forms (H2AsO4� and HAsO4
2�) atcircumneutral pH. It strongly adsorbs to metal oxide minerals, especially on iron(hydr)oxides (3). On the other hand, As(III) is more toxic and highly mobile since it takesa nonionic form (H3AsO3) under the neutral pH conditions found in most naturalenvironments (3–5). Contamination of drinking water by arsenic has caused serioushealth problems worldwide, particularly in Bangladesh and West Bengal (2).
Bacterial reduction of As(V) to As(III) plays a significant role in arsenic release fromanaerobic sediments into groundwater. Certain prokaryotes, such as Chrysiogenesarsenatis (6), Shewanella sp. strain ANA-3 (7, 8), and Bacillus selenitireducens (9), canutilize As(V) as a terminal electron acceptor for anaerobic growth. These dissimilatoryAs(V)-reducing bacteria have a gene sequence corresponding to respiratory As(V)reductase (Arr) in their genomes. Arr is a heterodimer protein belonging to thedimethyl sulfoxide (DMSO) reductase superfamily (10, 11), with two catalytic subunits.One of the subunits is ArrA, with a [4Fe-4S] center and molybdopterin as a cofactor. Theother is ArrB, with possibly three or four [4Fe-4S] clusters. While Arr is located in theperiplasm or outside surface of the cytoplasmic membrane, a wide variety of pro-karyotes possesses an unrelated cytoplasmic As(V) reductase (ArsC) for resistance ofarsenic inside the cells (12). The arsenic resistance system (Ars system) consists mainlyof ArsC, ArsR (a regulatory protein), ArsB or Acr3 [an As(III) efflux pump], ArsD (achaperone of the pump), and ArsA (a pump-driving ATPase). In dissimilatory As(V)-reducing bacteria, genes encoding ArrAB (arrAB) are sometimes flanked by the arsgenes, forming clusters of arsenic-metabolizing genes in their genomes (arsenic-metabolizing gene islands) (11, 13).
Krafft and Macy (6) found that the Arr activity of C. arsenatis under As(V)-respiringconditions was significantly higher than that under nitrate-respiring conditions. Theexpression dynamics of the arrA gene in Shewanella sp. ANA-3 were determined (14).The arrA gene of strain ANA-3 was expressed only under anaerobic conditions, while itwas repressed by other electron acceptors, such as oxygen, nitrate, and fumarate.Conversely, the arsC gene was expressed under both aerobic and anaerobic conditions.Transcriptional analysis revealed that the expression of arrA was induced by 0.1 �MAs(III), while 1,000 times more As(III) was required for the induction of arsC (14). Theeffect of arsenic, i.e., As(III) and As(V), on the expression of genes and proteins has alsobeen determined comprehensively in nondissimilatory As(V)-reducing bacteria such asPseudomonas aeruginosa (15), Caenibacter arsenoxydans (16), Herminiimonas arsenicoxy-dans (17), and Rhizobium sp. strain NT-26 (18). These studies suggest that the Arssystem proteins, as well as molecular chaperones, antioxidant proteins, and phosphatetransporters, were upregulated in the presence of As(III) or As(V).
In the last decade, molecular evidence on specific microorganisms involved in thereduction and subsequent mobilization of arsenic from anoxic sediments has beenenriched. The arrA gene and 16S rRNA gene closely related to the genus Geobacter havebeen detected frequently from arsenic-contaminated sediments of Bengal Delta (19,20), Mekong Delta (21), Cambodia (22), and paddy soil in China (23). Recently, Mirza etal. (24) designed a novel PCR primer pair for amplification of the arrA gene andevaluated the specificity of these new primers by high-throughput sequencing. Of the62,056 arrA gene sequences recovered from soil and groundwater samples, 16% werethought to be Geobacter-related phylotypes (cluster III), suggesting the importance ofthese bacteria in arsenic mobilization in nature.
To date, two strains of bacteria belonging to genus Geobacter, namely, G. uraniire-ducens Rf4 and G. lovleyi SZ, have been shown to possess arrAB genes in their genomes,but growth of these bacteria with As(V) as an electron acceptor has not been observed(25). Previously, a novel dissimilatory As(V)-reducing bacterium, Geobacter sp. strainOR-1, was isolated from Japanese rice paddy soil (26). Strain OR-1 also utilized fumarate,nitrate, soluble Fe(III), and ferrihydrite as electron acceptors and catalyzed dissolutionof arsenic from As(V)-adsorbed ferrihydrite. The draft genome of strain OR-1 containedtwo distinct arsenic islands (27) (Fig. 1). One included the arrAB genes, and they wereflanked by the ars genes. The other island consisted mainly of genes for the Ars system
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(Fig. 1). Geobacter sp. OR-1 is shown to be capable of As(V) respiration and is useful asa model microorganism that potentially impacts the mobilization of arsenic in floodedsoils and anoxic sediments. The aim of this study is to understand biochemical andmolecular mechanisms by which strain OR-1 copes with arsenic and to identify possibleenvironmental factors which trigger arsenic release by this strain. To this end, the activityand localization of Arr in strain OR-1 were determined, and the effect of arsenic on arrA andarsC gene expression was quantified by quantitative RT-PCR (qRT-PCR). In addition, com-prehensive proteomic analysis using cell extracts of cells grown with As(V) or fumarate asan electron acceptor was also performed by a combination of one-dimension SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and nano-liquid chromatography-tandemmass spectrometry (nano-LC-MS/MS).
RESULTS AND DISCUSSIONActivity and localization of Arr. For this study, strain OR-1 was grown anaerobi-
cally with 20 mM As(V) or fumarate as an electron acceptor. The enzyme activity of Arrin the crude extracts, obtained from such cells, was determined using reduced methylviologen (MV) as an electron donor. Although significant Arr activity (0.91 � 0.07 U mgprotein�1) was detected in fumarate-grown cells, 8.5 times higher activity (7.75 � 0.24U mg protein�1) was observed in As(V)-grown cells. To localize the Arr protein, theperiplasmic fraction was prepared from the washed whole cells by using the lysozyme-EDTA treatment. As shown in Table 1, 71% of the total Arr activity was found in theperiplasmic fraction. Malate dehydrogenase (MDH) activity, a cytoplasmic marker en-zyme, was found predominantly (82%) in the spheroplast fraction, indicating that theperiplasmic fraction was prepared appropriately. An apparent twin-arginine transloca-tion signal sequence (KRRDFLK) (28) was present in the N-terminal region of ArrA,suggesting its translocation to the periplasm. These results suggest that Arr of strainOR-1 is a periplasmic enzyme, as has been reported for Arr of other Gram-negativebacteria (6, 8).
Activity staining and LC-MS/MS analysis. After the periplasmic fraction was run onSDS-PAGE gels under mild denaturing conditions, the gel was stained for Arr activity
FIG 1 Two distinct arsenic-metabolizing gene islands found in the draft genome of Geobacter sp. OR-1 (GCA_000813145.1).(A) The arr island (locus tag OR1_RS06740 to OR1_RS06795) consists of arr genes (red), ars genes (blue), and molybdenumcofactor biosynthesis genes (green). Black arrows represent PCR primers designed for operon mapping. (B) The ars island (locustag OR1_RS02695 to OR1_RS02760) mainly consists of ars genes (blue) and putative tetrathionate reductase genes (ttr, yellow).Other genes, including universal stress protein genes (uspA), are represented in gray. HP, hypothetical protein.
TABLE 1 Localization of Arr protein in cell fractions of strain OR-1
Cell fraction
Mean � SD (relative %) ofa:
Total U of Arr Total mU of MDH
Crude extractsb 1.10 � 0.059 10.6 � 0.46Periplasm 0.655 � 0.13 (71.1) 1.63 � 0.022 (17.9)Spheroplasts 0.266 � 0.020 (28.9) 7.49 � 0.51 (82.1)an � 3.bThe specific activity of Arr in crude extracts was 7.75 U mg protein�1.
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using reduced MV as an electron donor. As shown in Fig. 2A, a single clear bandappeared on the gel, and it was excised and run again on SDS-PAGE gels under fullydenaturing conditions. As shown in Fig. 2B, two bands with apparent molecularweights of 120 and 30 kDa were observed after Coomassie brilliant blue (CBB) staining.Since the predicted molecular weight of ArrA in strain OR-1 was approximately 95 kDa,the band with higher molecular weight was excised, trypsin digested, and subjected toLC-MS/MS analysis. Five proteins with apparent molecular weights of 95 to 130 kDawere detected, and all of these proteins showed a protein identification probability of100% (Table 2). Among the five proteins, ArrA, which was annotated as molybdopterinoxidoreductase (NCBI RefSeq accession no. WP_041970871.1), showed exclusive uniquepeptide counts of 8 and sequence coverage of 10%. Other proteins included ahypothetical protein, pyruvate:ferredoxin oxidoreductase, and pyruvate carboxylase,with sequence coverages of 2.7% to 21%. Interestingly, another molybdopterin oxi-doreductase (NCBI RefSeq accession no. WP_041969781.1) showed the highest exclu-sive unique peptide count of 65 and sequence coverage of 66%. Due to its relativelyclose relationship with tetrathionate reductase subunit A of Salmonella enterica (see Fig.S1 in the supplemental material), this protein is referred to here as TtrA.
arr and ars islands. As shown in Fig. 1, the draft genome of strain OR-1 containedtwo distinct arsenic-metabolizing gene islands. One of the two islands, the arr island,included arrAB genes, and they were flanked by genes for a TorD-like chaperone (arrD,which might be involved in cofactor insertion into ArrA) and a putative Fe-S protein(arrE) (29). Notably, however, the gene encoding a membrane-integral protein (arrC)
FIG 2 (A) Activity staining of Arr protein. The concentrated periplasmic fraction was denatured partially(2% SDS and 5% 2-mercaptoethanol) on ice for 5 min and run on sodium dodecyl sulfate-polyacrylamidegel electrophoresis gels at 4°C. After the electrophoresis, the gel was stained anaerobically with reducedmethyl viologen and arsenate as the electron donor and acceptor, respectively. (B) Separation of theexcised active band under completely denatured conditions (100°C, 5 min). The gel was stained withCoomassie brilliant blue R-250. Lane 1, standard marker proteins. The band with molecular weight ofapproximately 120 kDa (lane 2) was excised, trypsin digested, and subjected to liquid chromatography-tandem mass spectrometry analysis (see Table 2).
TABLE 2 LC-MS/MS identification of proteins recovered from the active banda
Protein name GenBank accession no.Predictedmolecular wt (Da)
Exclusive uniquepeptide count
Sequencecoverage (%)
Molybdopterin oxidoreductase (TtrA) WP_041969781.1 119,759 65 65.5Hypothetical protein WP_041972467.1 104,300 18 21.2Molybdopterin oxidoreductase (ArrA) WP_041970871.1 94,845 8 10.0Pyruvate:ferredoxin oxidoreductase WP_041973721.1 129,875 6 6.62Pyruvate carboxylase WP_082002763 125,469 2 2.72aFor all proteins, the protein identification probability was 100%.
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was absent. In the same island, several ars genes (arsR, acr3, arsA, and arsD) werelocated adjacent to the arrA gene, together with several molybdenum cofactor bio-synthesis genes (mobA, moeA, moaA, and mosC). The second arsenic-metabolizing geneisland (ars island) consisted of a series of ars genes, such as arsR, arsC, acr3, arsA, andarsD, and genes encoding universal stress protein (uspA). Interestingly, genes encodingtwo catalytic subunits of putative tetrathionate reductase (ttrAB), whose translationalproduct was detected in LC-MS/MS analysis of the active band, were located adjacentto the ars island (Fig. 1).
The gene arrangement of arrA-arrB-arrD-arrE in the arr island is conserved not onlyin strain OR-1 but also in G. lovleyi SZ and G. uraniireducens Rf4 (29). To test whetherthese genes are cotranscribed as a single operon, PCR primers for reverse transcription-PCR (RT-PCR) were designed spanning across arrB and arrA (RT1), arrA and arsR (RT2),arrD and arrB (RT3), and arrE and arrD (RT4) (Fig. 1). Total RNA was extracted from cellsgrown on As(V) and fumarate, and cDNA was synthesized. PCR products were observedin primer pairs RT1, RT3, and RT4, but not in RT2 (Fig. S2). These results suggest thatarrA, arrB, arrD, and arrE are transcribed together into a single mRNA, while arrA andarsR are not cotranscribed.
Transcriptional analysis of arrA, arsC, and ttrA genes. The expression of arrA andarsC genes relative to that of the housekeeping gene recA was quantified and com-pared by using RNA extracted from As(V)- or fumarate-grown cells. As shown in Table3, the arrA gene in As(V)-grown cells showed a 51-fold upregulation compared to thatin fumarate-grown cells (0.77 versus 0.015). Similarly, arsC gene expression in As(V)-grown cells increased 305-fold compared to that in fumarate-grown cells (0.58 versus0.0019). A similar observation has been reported in Shewanella sp. ANA-3, whichexhibited a higher abundance of arrA and arsC transcripts in As(V)-grown cells than inthose grown with other electron acceptors (14). The expression of the putative ttrAgene, which was located adjacent to the ars island, was also quantified. Although thettrA gene was expressed substantially in fumarate-grown cells, it was upregulated only6.7-fold in As(V)-grown cells (1.0 versus 0.15).
In Shewanella sp. ANA-3, both arrA and arsC genes were induced mainly by As(III)but not by As(V) (14). Thus, effect of As(III) on the expression of arrA and arsC genes instrain OR-1 was determined. To do so, the strain was grown on fumarate as the electronacceptor in the absence or presence of 1 nM (0.001 �M) to 1 mM (1,000 �M) As(III). Asshown in Fig. 3, the expression of arrA gene was detected at 1 �M As(III), and itsexpression increased up to 22-fold compared to the no-As(III) control. In contrast, theexpression of arsC gene required a 500 times higher concentration of As(III) than thatrequired for arrA, and an increase of up to 69-fold was detected compared to itsexpression in a no-As(III) control. Similar experiments were performed with the ttrAgene using As(III) concentrations of 0 to 100 �M. The expression of the ttrA gene wasdetected even in the absence of As(III), but its expression increased up to 5-foldcompared to that in a no-As(III) control (Fig. 3).
The induction profile of arrA and arsC gene expression in strain OR-1 was similar tothat in Shewanella sp. ANA-3, whose arsC required 1,000 times more As(III) for itsinduction than arrA (14). In strain ANA-3, not only As(III), but As(V) also induced arr geneexpression, although its induction level was 10-fold lower than that of As(III) (14).
TABLE 3 Expression of arrA, arsC, and ttrA genes relative to that of recA gene in As(V)- orfumarate-grown cells
Gene
Mean � SD quantity ratios by growth conditiona
As(V) Fumarate
arrA 0.77 � 0.082 0.015 � 0.0052arsC 0.58 � 0.16 0.0019 � 0.00050ttrA 1.0 � 0.012 0.15 � 0.080aValues represent the ratio of the relative quantity of arrA, arsC, or ttrA transcripts to that of thehousekeeping gene recA transcripts. n � 3.
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However, it is still unclear whether As(V) has the same impact on arr gene expressionin strain OR-1.
Comparative proteomic analysis of strain OR-1 grown on As(V) or fumarate. Inthe proteomic analysis, a total of 1,203 proteins with at least two unique peptides wereidentified, and they corresponded to 29% of the predicted coding sequence (CDS) ofstrain OR-1. Among these, about half of the proteins (613 proteins) were detected inboth As(V)- and fumarate-grown cells, and they are summarized in Table S1. In contrast,specific induction of 372 and 218 proteins was observed in As(V)- and fumarate-growncells, respectively (Tables S2 and S3). To date, the effect of arsenic stress on the proteinexpression profile has been determined in various aerobic bacteria (15–18, 30–33). Thisstudy reports comprehensive proteomic analysis performed with an anaerobic As(V)-respiring bacterium. In the following sections, a detailed description of selected pro-teins will be presented, whose expression profiles appeared to be significantly affectedunder the As(V)-respiring conditions used in this study.
Arr and Ars proteins. As shown in Table 4, ArrA, ArrB, and ArrD were identified inAs(V)-grown cells but were absent in fumarate-grown cells. In particular, ArrA and ArrBwere expressed in high abundance, accounting for 0.76% and 1.1% of total expressedproteins, respectively. As described above, ArrA peptide sequences were actuallyidentified in the SDS-PAGE band stained for dissimilatory As(V) reductase activity (Table2). These results suggest that Arr is indispensable and directly involved in dissimilatoryAs(V) reduction by strain OR-1.
In As(V)-grown cells, two ArsA proteins were found to be expressed in higherabundance (Table 4). Recently, Dang et al. (34) found that both ArsC and Acr3 proteinswere important in G. sulfurreducens under arsenic stress, since an acr3-null mutant wasnot able to grow in the presence of either As(V) or As(III), while an arsC-null mutantcould grow in the presence of As(III) but not As(V). Although neither ArsC nor Acr3 wasidentified in the proteomic analysis, arsC gene expression was upregulated in responseto arsenic exposure (Table 3 and Fig. 3). Therefore, it would be reasonable to considerthat the Arr system as well as the Ars resistance system play a pivotal role underAs(V)-respiring conditions. It is possible that the relatively low molecular weight of ArsC(15 kDa) prevented its detection.
FIG 3 Effect of increasing concentrations of arsenite [As(III)] on expression of arrA, arsC, and ttrA genes.The expression of arrA, arsC, and ttrA genes relative to that of the recA gene was determined as a functionof increasing As(III) concentrations in cultures of Geobacter sp. OR-1. The strain was grown anaerobicallywith fumarate plus the indicated concentrations of As(III). Symbols represent the mean values obtainedfor triplicate determinations, and bars indicate standard deviations.
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Ttr proteins. Both TtrA and TtrB proteins were identified in the proteomics analysis,and they were found to have accumulated in As(V)-grown cells (Table 4). The resultsfrom transcriptional and proteomics analyses imply that Ttr plays some roles in dis-similatory As(V) reduction by strain OR-1. TtrA is one of the DMSO reductase super-family proteins and is phylogenetically closely related with ArrA (Fig. S1). Although itwas genetically characterized in S. enterica as a protein essential for anaerobic tetra-thionate (S4O6
2�) respiration (35, 36), TtrA-like proteins may also be involved indissimilatory As(V) reduction in archaea such as Pyrobaculum aerophilum and Pyrobacu-lum calidifontis (37). In addition, TtrA is one of candidates for respiratory As(V) reductasein dissimilatory As(V)-reducing bacteria, such as Melioribacter roseus (38) and Citrobactersp. strain TSA-1 (39), both of which lack homologs of arrA in their genomes. Thus, itmight be possible that TtrA acts as an auxiliary As(V) reductase in strain OR-1. Consid-ering that low but significant As(V)-reducing activity was still observed in cells grownwithout As(V), TtrA could participate in the background level of As(V) reductase activityin strain OR-1.
Proteins involved in phosphate transport and molybdenum cofactor biosyn-thesis. Bacteria take up As(V) through phosphate transporters as it is a structural analogof phosphate. In Escherichia coli, the low-affinity phosphate transport system (Pit)contributes mainly to As(V) uptake (40, 41). In the proteomic analysis carried out in thisstudy, three types of high-affinity phosphate ABC transporter substrate-binding protein(PstS) were identified, of which two proteins were found to have accumulated inAs(V)-grown cells (Table S4). Generally, the bacterial Pst system belongs to the Phoregulon and induced when the external phosphate level is lower than 4 �M (42). Inaddition, Kang et al. (43) reported that As(III) upregulated the expression of PstS underlow-phosphate conditions in Agrobacterium tumefaciens 5A, an As(III)-oxidizing bacte-rium. Thus, it might be possible that low levels of phosphate and As(III) that accumu-lated in the medium induced the expression of PstS proteins during the growth ofstrain OR-1 on As(V). Upregulation of the Pst system may indicate facilitation of specificphosphate uptake in the presence of high levels of As(V) while avoiding As(V) entry intothe cells through the Pit system.
MobA, MoeA, and MoaA, all of which are encoded by the arr island, were identifiedspecifically in As(V)-grown cells (Table 4). In addition, other molybdenum cofactorbiosynthesis proteins that are not encoded by the arr island were also identified,among which two proteins (MobB and MoeA) were found to have accumulated inAs(V)-grown cells (Table S4). Accumulation of these proteins may occur concurrentlywith the upregulation of ArrA biosynthesis, since it contains a molybdenum cofactor inits active site.
TABLE 4 arr and ars island proteins expressed in cells of strain OR-1 identified in proteomic analysis
NCBI RefSeq accession no.by protein locus Gene name Protein name
Antioxidant proteins. Arsenic is known to generate reactive oxygen species (ROS),such as superoxide anion, hydroxyl radical, hydrogen peroxide, singlet oxygen, andperoxyl radicals (44). Thus, arsenic stress triggers the expression of antioxidant proteins,including catalase, superoxide dismutase (SOD), thioredoxin, peroxiredoxin, and gluta-thione reductase in many bacteria (15, 18, 31–33). Analysis of the draft genome of strainOR-1 revealed that antioxidant proteins, such as SOD, glutathione peroxidase, thiolperoxidase, peroxiredoxin, and rubrerythrin, but not catalase-peroxidase, were encodedin it (27). However, only limited numbers of ROS-scavenging proteins were identified inthe proteomics analysis done here, while only rubrerythrin and peroxiredoxin werefound to have accumulated in As(V)-grown cells (Table S4). Although it remainspossible that some of the antioxidant proteins were not detected due to their lowmolecular weights, these results suggest that known antioxidant proteins play onlyminor roles during As(V) respiration by strain OR-1. Under the strictly anaerobicconditions used in this study, ROS formation might have been repressed considerablycompared to that observed under aerobic conditions.
Proteins involved in folding, assembly, and quality control. In contrast toantioxidant proteins, a wide variety of proteins involved in protein folding, assembly,and quality control were found to be upregulated under the As(V)-respiring conditions(Table S4). In particular, members of the Hsp60, Hsp70, and Hsp90 systems (DnaK, DnaJ,GrpE, GroEL, and HtpG), ClpB, and trigger factor, all of which are involved in disaggre-gation and refolding of denatured polypeptides, were more abundant in As(V)-growncells. Accumulation of such proteins under arsenic stress has also been observed inother bacteria (15, 17, 30, 32, 33). Similarly, proteins involved in the degradation ofmisfolded proteins (ClpX, Lon, and DegQ proteases) were expressed at a higherabundance in As(V)-grown cells. These results suggest that proteins oxidatively dam-aged by arsenic and/or ROS are rescued by stress-induced multichaperone systems, butterminally misfolded proteins are degraded before they can form toxic aggregates.Accumulation of a chaperone for outer membrane proteins (SurA) and a periplasmicdisulfide isomerase (DsbC) in As(V)-grown cells indicates that not only cytoplasmic butalso secreted proteins, such as Arr, are among the targeted misfolded proteins. Asshown in Table S5, GroEL, DnaK, and HtpG were among the top 10 most highlyexpressed proteins in As(V)-grown cells. While GroEL is also one of the most highlyexpressed proteins in fumarate-grown cells, DnaK and HtpG are not.
Proteins involved in central carbon metabolism. Acetate permease and tricar-boxylic acid (TCA) cycle enzymes, such as isocitrate dehydrogenase, 2-oxoacid:ferre-doxin oxidoreductase, fumarate reductase/succinate dehydrogenase, and malate de-hydrogenase, are some of the other most highly expressed proteins in cells of strainOR-1 (Table S5). This indicates that the TCA cycle is very important for energy conser-vation by strain OR-1 when it is grown on acetate as the carbon source. Although in thepresence of arsenic TCA cycle enzymes seemed to be governed by a mixed regulatorypattern, several key enzymes involved in anaplerosis and gluconeogenesis, such aspyruvate carboxylase (59.3-fold), phosphoenolpyruvate carboxykinase (GTP) (28.1-fold),pyruvate phosphate dikinase (18.2-fold), and fructose-bisphosphatase (8.7-fold), wereexpressed at a higher abundance in As(V)-grown cells (Table S4). Several acetate-activating enzymes involved in the formation of acetyl-coenzyme A (acetyl-CoA),including acetate kinase/phosphotransacetylase, acetyl-CoA hydrolase/transferase, andacetyl-CoA synthetase, were also accumulated in As(V)-grown cells (Table S4).
As(III) is known to inhibit pyruvate dehydrogenase and 2-oxoglutarate dehydroge-nase multienzyme complexes since it binds to the lipoic acid and dithiol moieties ofthese enzymes (45–48). In addition, ROS generated by arsenic degrades Fe-S clusters,which are components of aconitase, succinate dehydrogenase, and fumarase. Althoughpyruvate dehydrogenase may not operate significantly in Geobacter spp. grown onacetate (49–51), it is possible that TCA cycle in the strain OR-1 is, at least in part,defective during As(V) respiration. In several bacteria, it is known that As(III) significantlyinhibited the TCA cycle and resulted in a shift in carbon metabolism from the citric acid
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pathway to the glyoxylate pathway in order to compensate for the enzymes disruptedby As(III) (47, 52, 53). However, strain OR-1 lacks key enzymes of the glyoxylate pathway(isocitrate lyase and malate synthase) and is unable to bypass the TCA cycle for carryingout anaplerosis and gluconeogenesis. To overcome this limitation, strain OR-1 mayincrease levels of pyruvate carboxylase, a key anaplerotic enzyme catalyzing pyruvateconversion to oxaloacetate (Table S4). It seems likely that strain OR-1 copes with arsenicstress by providing the TCA cycle with acetyl-CoA and oxaloacetate for sustainableenergy production while promoting gluconeogenesis for biomass production. A pos-sible key intermediate is pyruvate produced from acetyl-CoA by pyruvate:ferredoxinoxidoreductase (49).
Gluconeogenesis is not the only way for biomass production. Especially, glutamateis one of important building blocks and should be produced from acetate even in thepresence of arsenic. As shown in Table S4 (arginine biosynthesis), NADP-specificglutamate dehydrogenase was expressed at a higher abundance in As(V)-grown cells.In addition, two types of putative pyridoxal phosphate-dependent aspartate amino-transferase (YhdR and AspC), which transform aspartate and 2-oxoglutarate to oxalo-acetate and glutamate, were found to have accumulated in As(V)-grown cells. Theseresults suggest that glutamate biosynthesis actually occurred under As(V)-respiringconditions.
Proteins involved in DNA replication and repair. Arsenic can cause DNA damage,such as single- or double-strand DNA breaks and inhibition of DNA repair machinery(44). Corresponding with this, various proteins involved in DNA replication and repairwere found to be accumulated in As(V)-grown cells (Table S4). These included DNApolymerase I, DNA ligase, DNA gyrase, DNA glycosylase, and proteins involved in DNAdamage recognition (UvrA and UvrC), methyl-directed DNA mismatch repair (MutS andMutL), and recombinational repair of double-strand DNA breaks (RecA, RecB, and RadA).In addition, a class II ribonucleotide reductase (NrdJ), which converts ribonucleotides(NTPs) to deoxyribonucleotides (dNTPs) as building blocks for DNA replication andrepair, was expressed at high abundance (3.2% of total protein) in As(V)-grown cells.These results suggest that significant DNA lesions may occur during As(V) respiration,but multiple mechanisms leading to DNA repair cooperate in As(V)-grown cells, whichare probably maintained by a sufficient pool of dNTPs.
Regulatory proteins. The draft genome of strain OR-1 contained at least five arsR
homologues, among which two genes were located within the arr and ars islands (Fig.1). However, neither of these gene products (ArsR) was identified in the proteomicanalysis, possibly due to their low molecular weights or low expression levels. In E. coli,ArsR is known to repress the transcription of ars genes in the absence of As(III) butderepress ars operon genes in the presence of As(III) (54). In Shewanella sp. ANA-3, anArsR protein (ArsR2) coregulated the arr and ars genes (55). Recently, it was alsoreported that an ArsR protein (ArsR1) regulated the transcription of the ars operon inG. sulfurreducens (34). Each arsenic island had its own arsR gene in strain OR-1, and itis tempting to speculate that the arr and ars islands are regulated separately byrespective ArsR proteins. The different induction profile of arrA and arsC genes by As(III)observed in this study might reflect different regulatory proteins involved in theexpression of these genes. Kang et al. (56) reported that multiple arsR genes inAgrobacterium tumefaciens 5A exhibited sensitivity to As(III) but displayed a range ofexpression levels that differed as a function of the regulatory background.
Four putative sigma factors were also identified in the proteomic analysis done inthis study, among which homologues of RpoS (�S) and RpoH were identified only inAs(V)-grown cells (Table S4). In G. sulfurreducens, an RpoS homologue was reported tobe a global regulator of gene expression, and the RpoS-dependent genes includedthose involved in TCA cycle, signal transduction, protein synthesis and degradation,and amino acid metabolism (57). In contrast, an rpoH homologue gene of G. sulfurre-ducens was induced by a heat shock, and genes such as grpE, dnaK, groES, and htpGwere inducible in an rpoH-dependent manner (58). Since these gene products or
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related proteins (GroEL) were found to have accumulated in As(V)-grown cells, RpoHmay also play some roles in strain OR-1 under As(V)-respiring conditions. In As(III)-oxidizing bacteria such as H. arsenicoxydans and Agrobacterium tumefaciens, RpoN (�54)was reported to be involved in the control of As(III) oxidase gene (aioAB) expression (59,60). As a first step in clarifying the regulatory networks of strain OR-1 during As(V)respiration, quantification of arsR genes and sigma factor genes transcripts is underway.
Other proteins. Other proteins, whose expression tended to be upregulated in thisstudy on As(V)-grown cells, included those involved in aminoacyl-tRNA biosynthesis,translation elongation factors, amino acid biosynthesis and metabolism, purine andpyrimidine metabolism, fatty acid biosynthesis, and peptidoglycan biosynthesis (TableS4). On the other hand, proteins involved in energy production (ATP synthase), che-motaxis, twitching motility, and protein secretion seemed to be downregulated inAs(V)-grown cells (Table S4).
To maintain cellular homeostasis under arsenic stress, the damaged proteins mustquickly be renewed again. Upregulation of amino acid biosynthesis, aminoacyl-tRNAbiosynthesis, translation elongation factors, and proteins involved in posttranslationalmodification (molybdenum cofactor biosynthesis proteins, molecular chaperones, andpeptidyl-prolyl isomerases) observed in this study suggests that protein synthesis mayoccur during As(V) respiration. Similarly, the observed upregulation of purine andpyrimidine metabolism may reflect nucleotide synthesis under As(V)-respiring condi-tions. Accumulation of fatty acid biosynthesis proteins might indicate the restoration oflipid peroxidation caused by ROS or the modification of cell membrane composition torepress the entry of arsenic into the cells.
Conclusion. In this study, it was demonstrated that Arr is directly involved indissimilatory As(V) reduction by Geobacter sp. strain OR-1. In addition, Arr was upregu-lated as indicated by increased mRNA and protein levels under As(V)-respiring condi-tions. Furthermore, various proteins that are differentially expressed in cells of strainOR-1 in the presence of arsenic were identified. The results obtained in this studysuggest that strain OR-1 copes with arsenic stress by upregulating the proteins involvedin the following processes: (i) the conventional Ars system for reduction and extrusionof arsenic out of the cells by an efflux pump; (ii) protein folding, assembly, and qualitycontrol for rescue of oxidatively damaged proteins and degradation of terminallymisfolded proteins; (iii) DNA replication and repair for the restoration of single- anddouble-strand DNA breaks; (iv) anaplerosis and gluconeogenesis pathways for sustain-able energy production and biomass formation; and (v) protein and nucleotide syn-thesis for the renewal of damaged proteins and nucleotides. Interestingly, many ofthese metabolic processes have been reported to be upregulated in other bacteriaunder arsenic stress (61), suggesting that mechanisms by which bacteria copes withthis toxic metalloid are conserved not only in aerobic bacteria but also in anaerobicAs(V) reducers. Considering that dissimilatory As(V)-reducing bacteria are involved inarsenic release from anaerobic sediments worldwide, such information is important inassessing and considering possible countermeasures for the prevention of microbialarsenic release in nature. Furthermore, dissimilatory As(V)-reducing bacteria are re-cently being considered as potential candidates for use in the restoration of arsenic-contaminated soils, since they can extract and release arsenic from the soils into theaqueous phase, lowering the soil arsenic concentration to levels below the acceptedstandards (62, 63). Thus, the results obtained in this study could provide importantclues for precise control of dissimilatory As(V)-reducing bacteria at the sites of biore-mediation.
MATERIALS AND METHODSBacterial strain and growth conditions. Strain OR-1 was cultured at 30°C in a minimal medium (26).
Strict anaerobic technique (64) was used in the preparation of the minimal medium and the manipu-lation of the cultures. The medium was dispensed into 60-ml serum bottles under an N2:CO2 (80:20)atmosphere and autoclaved. The medium contained the following (per liter): NH4Cl (0.535 g), KH2PO4
(0.136 g), MgCl2·6H2O (0.204 g), CaCl2·2H2O (0.147 g), cysteine-HCl (0.176 g), trace mineral element
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solution (1 ml), vitamin solution (1 ml), selenite-tungstate solution (1 ml), and NaHCO3 (2.52 g). Acetate(10 mM) was used as an electron donor, and As(V) (20 mM as K2HAsO4) or fumarate (20 mM) was usedas an electron acceptor. In experiments where the effect of As(III) on the expression of different geneswas determined, the strain was grown on 20 mM fumarate with various concentrations of As(III), i.e., 1 nM(0.001 �M) to 1 mM (1,000 �M) as NaAsO2. Acetate, As(V), As(III), fumarate, and cysteine-HCl were addedseparately from sterile anaerobic stock solutions.
Preparation and fractionation of crude cell extracts. For the preparation of the crude extracts,cells grown on As(V) or fumarate were collected, washed, and resuspended in 20 mM Tris-HCl (pH 6.8).They were disrupted by sonication (Q500 sonicator; Qsonica, Newtown, CT, USA) at 20 kHz and 30%amplitude for 2 min, followed by centrifugation (20,000 � g, 30 min, 4°C) to remove cell debris. Theperiplasmic fraction was prepared according to the method of Davidson and Sun (65). Briefly, cells wereresuspended in 200 mM Tris-HCl (pH 7.5) containing 0.5 mM Na2EDTA, 0.35 M sucrose, and 3 mg ml�1
lysozyme. After incubation at 30°C for 30 min, an equal volume of distilled water was added to inducea mild osmotic shock. This suspension was incubated on ice for 2 h, and using centrifugation (20,000 � g,30 min, 4°C), the periplasmic fraction was separated from the spheroplasts and any unbroken cells. Thedisruption of spheroplasts and removal of cell debris were carried out as described above.
Enzyme assay. Arr activity was assayed spectrophotometrically in a sealed quartz cuvette at 25°C bymonitoring the oxidation of reduced methyl viologen (MV) (�578 � 9.7 mM�1 cm�1) as an electron donor.The reaction mixture (0.7 ml) contained 20 mM Tris-HCl (pH 6.8), 0.3 mM MV, 10 mM As(V), and anappropriate amount of the crude enzyme. After the reaction mixture was degassed and sparged with N2
gas, the reaction was started by the addition of a small amount of sodium dithionite to give anabsorbance at 578 nm of 1.5 to 2.0. One unit (U) of the reductase activity was defined as the amount ofenzyme protein required to oxidize 1 �mol reduced MV per minute. The activities were calculated frominitial rates of oxidation of MV, and all measurements were corrected for nonenzymatic oxidation of MVby subtracting the rates of control samples, in which enzyme was omitted from the reaction mixture.Protein concentration was determined by Protein assay kit (Bio-Rad) with bovine serum albumin as astandard protein. MDH activity was measured spectrophotometrically by monitoring the rate of disap-pearance of NADH (66). The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.1 mM NADH (�340 �6.22 mM�1 cm�1), 0.2 mM oxaloacetate, and an appropriate amount of the crude enzyme.
Electrophoresis, activity staining, and LC-MS/MS analyses. The periplasmic fraction prepared asdescribed above was concentrated by ultrafiltration (Amicon Ultra 10K centrifugal filter; Millipore,Bedford, MA, USA). To separate proteins included in the periplasmic fraction, SDS-PAGE was carried outin two steps. In the first step, partially denatured samples (2% SDS and 5% 2-mercaptoethanol on ice for5 min) were used for electrophoresis performed at 4°C using 6% polyacrylamide gel in 25 mM Tris-glycinebuffer (pH 8.3) by the method described by Laemmli (67). Following this electrophoresis, the gel wasincubated under nitrogen atmosphere with 20 mM Tris-HCl (pH 6.8) containing 0.3 mM MV, 10 mM As(V),and 6 mM dithionite. In the second step, the proteins from the clear band (active band) whichappeared on the gel were excised, boiled for complete denaturation (with 2% SDS and 5%2-mercaptoethanol for 5 min), and then run through SDS-PAGE again. A protein molecular weightmarker (TaKaRa, Otsu, Japan) was used as standard marker proteins. Proteins were visualized bystaining with CBB R-250, and the selected band was excised, trypsin digested, and subjected toLC-MS/MS analysis, as described previously (68).
Transcriptional analysis. RNA was prepared from triplicate cultures for all growth conditions, i.e., onAs(V), fumarate, or fumarate plus 1 nM (0.001 �M) to 1 mM (1,000 �M) As(III). Cells at the late-exponentialphase of growth (1.5 days after inoculation) were collected by centrifugation (10,000 � g for 10 min at4°C), and pellets were immediately frozen in a liquid nitrogen bath and stored at �80°C. The total RNAwas extracted with the RNeasy miniprep kit (Qiagen, Hilden, Germany) according to the protocolprovided by the manufacturer. The eluents were then treated with the Turbo DNA-free kit (Ambion,Carlsbad, CA, USA) to remove residual DNA. To ensure that RNA samples were not contaminated withgenomic DNA, PCR amplification with primers targeting the 16S rRNA gene (338F and 518R) (69) wasconducted. The DNase-treated RNA samples were stored at – 80°C until use. All RNA samples hadA260/A280 ratios of 1.8 to 2.0, indicating their high purity.
cDNA was synthesized using a SuperScript first-strand synthesis system for RT-PCR (Invitrogen,Carlsbad, CA, USA), in which RNA samples were primed by random hexamers through reverse transcrip-tion. To 11 �l of RNA samples, 1 �l of a 10 mM dinucleoside triphosphate (dNTP) mixture (Invitrogen) and1 �l of random hexamers (Invitrogen) were added. The mixture was incubated at 65°C for 5 min. Aftercooling in ice for at least 1 min, 4 �l of 5 � SuperScript IV buffer, 1 �l of 0.1 M dithiothreitol, 1 �l ofRNaseOUT solution (Invitrogen), and 1 �l of SuperScript IV reverse transcriptase (Invitrogen) were added.The reaction mixture was incubated with the following temperature cycle: 10 min at 23°C, 10 min at 50°C,and 10 min at 80°C. A control without reverse transcriptase was included for each RNA sample to ensurethat DNA contamination had no effect on mRNA detection. The triplicate samples were treatedindependently through RNA extraction and cDNA synthesis and yielded three separate cDNA samplesthat were later subjected to quantitative PCR (qPCR).
Mapping of the arr genes using RNA transcript was performed on total RNA extracted from cellsgrown on As(V) or fumarate. After cDNA was synthesized as described above, PCRs were performed withvarious combinations of primers (Table 5) to determine whether arr genes are cotranscribed as a singleoperon. The primers were designed using the Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). Thereaction profile was as follows: 95°C for 10 min, 30 cycles of 95°C for 30 s, 60°C for 45 s, and 72°C for 1 min,and then 72°C for 7 min.
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qRT-PCR. Quantification of arrA, arsC, and ttrA genes in the cDNA samples were performed withqPCR. The housekeeping gene recA was also quantified in the cDNA samples for normalization of theqRT-PCR data, since the transcription of this gene was reported to be constitutive in Geobactersulfurreducens (70). The gene expression levels were presented as the number of arrA, arsC, or ttrA genetranscripts per the number of recA transcripts. As shown in Table 6, the new primers were designed forqPCR, and amplicon sizes were limited to �150 bp for accurate quantification. SYBR green detectionchemistry was used for qPCR assays by using an ABI Prism 7000 instrument (Applied Biosystems). The25-�l qRT-PCR mixture contained 12.5 �l of Power SYBR green PCR master mix (Applied Biosystems), 0.5�l of a 10 �M concentration of each primer, 2 �l diluted template (cDNA), and 9.5 �l of nuclease-freewater. The reaction profile was as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min.The amplification reactions were run at least in triplicate. Amplification of the no-template controls andDNase-treated RNA samples yielded negative results. A final dissociation step was performed to obtainthe melting curves (thermal profile) of the amplicons, and they indicated the target specificity of theqPCRs. Standard curves for arrA, arsC, ttrA, and recA genes were constructed with serial dilutions (thatcovered a range of 5 orders of magnitude) of cDNA samples prepared from the cells grown on As(V).Standard curves were generated by plotting the cycle threshold (CT) values as a function of the log of theamount of cDNA. The slopes of four standard curves were used for calculation of the PCR efficiencies (E)according to the equation
E�%� � [10��1⁄slope� – 1] � 100
The E values obtained for all genes and primer pairs ranged from 90% to 110%, with R2 values of 0.98to 0.99 (Table 6).
Proteomic analysis. Cells grown on As(V) or fumarate as the electron acceptor were harvested atearly stationary phase by centrifugation (6,000 � g, 15 min, 4°C) and washed twice with 8% NaCl. Theharvested cells were lysed with the ReadyPrep protein extraction kit (Bio-Rad, Hercules, CA, USA). Totalproteins (50 �g) were separated using 10% SDS-PAGE gels (90 mm by 85 mm) and stained with CBBG-250 (Fig. S3). The gel lanes were cut into 45 1-mm-long strips. The gel strips were completely destainedwith 30% acetonitrile (ACN) in 25 mM NH4HCO3, reduced with 10 mM dithiothreitol, and alkylated with55 mM iodoacetamide. After these gel strips dried completely, in-gel digestion was performed with 40 �lof sequencing-grade modified trypsin (12.5 �g ml�1 in 50 mM NH4HCO3) at 37°C overnight. The digestedpeptides were extracted with 25 mM NH4HCO3 in 60% ACN and further extracted twice with 5% formicacid in 70% ACN.
LC-MS/MS analysis was performed using an LTQ ion-trap MS (Thermo Fisher Scientific, Waltham, MA,USA) coupled with a Paradigm MS2 multidimensional high-performance liquid chromatogram (HPLC;AMR, Tokyo, Japan) and a nanospray electrospray ionization device (Michrom Bioresources, Auburn, CA,USA). The tryptic peptides were loaded onto an L-column2 ODS (Chemicals Evaluation and ResearchInstitute, Tokyo, Japan) packed with C18 modified silica particles (5 �m, 12-nm pore size) and wereseparated by a linear gradient of 15 to 65% buffer B for 40 min, followed by a gradient of 65 to 95% bufferB for 1 min (buffer A, 2% methanol and 0.1% formic acid in H2O; buffer B, 90% methanol and 0.1% formicacid in H2O) at a flow rate of 1 �l min�1. Peptide spectra were recorded in a mass range of m/z 450 to1,800. MS/MS spectra were acquired in data-dependent scan mode. After the full spectrum scan, 1 MS/MS
TABLE 5 PCR primers for operon mapping
Primer pair Name Sequence (5=–3=)RT1 arrA-1 GGTTTGCCAACCTGTA
spectrum of the single most intense peaks was also collected. The dynamic exclusion features were setas follows: a repeat count of 1 within 30 s, an exclusion duration of 180 s, and an exclusion list size of 50.The obtained MS/MS data were searched against data on the strain OR-1 in the NCBI (NCBI RefSeqaccession no. NZ_BAZF00000000) using the Mascot program version 2.5 (Matrix Science, London, UK) onan in-house server to identify proteins. Search parameters were set as follows: tryptic digestion with amaximum of 2 missed cleavage sites; fixed modifications, carbamidomethyl cysteine; variable modifica-tions, methionine oxidation; peptide masses, monoisotopic, positive charge (�1, �2, �3) of peptide; andmass tolerance of 1.2 Da for the precursor ion and 0.8 Da for product ions. To assess false-positiveidentifications, an automatic decoy search was performed against a randomized database with a defaultsignificance threshold of P � 0.05; the false-discovery rate at the identity threshold was below 6.9%.
The protein abundance index (PAI) was defined as: PAI � Nobserved/Nobservable, where Nobserved andNobservable are the number of experimentally observed peptides and the number of theoretically observ-able peptides for each protein, respectively (71, 72). Label-free quantitative analysis of the abundance ofthe identified proteins was performed using the exponentially modified PAI (emPAI) values that areprovided by the Mascot program, as follows: emPAI � 10PAI � 1.
Relative protein content in molar fraction percentage was then determined using the followingequation: relative protein content (mol %) � emPAI/� (emPAI) � 100, where � (emPAI) is the summationof the emPAI values for all of the identified proteins (71, 72).
SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM
ACKNOWLEDGMENTSThis work was financially supported by JSPS KAKENHI grant 26450086.We thank T. Kataoka (Fukui Prefectural University) for technical assistance, C. M.
Yeager, S. Iyer, and T. Sanchez (Los Alamos National Laboratory) for preliminaryproteomic analysis. S.A. also would like to thank S. Yamamura (National Institute ofEnvironmental Studies) for helpful discussion.
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