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

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.

ABSTRACT

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.

INTRODUCTION

Arsenic, a toxic metalloid, is released into the environment from both natural and anthropogenic sources. In addition to the weathering, oxidation, and erosion of sulfide minerals, arsenic can be mobilized through anthropogenic activities such as mining operations, smelting, and coal combustion (1, 2). The predominant forms of arsenic in soils and aquifers are inorganic arsenate [As(V)] and arsenite [As(III)] (1–3). As(V) is thermodynamically stable under oxic conditions, whereas As(III) is stable under reducing conditions. As(III) exhibits higher toxicity than As(V). Generally, As(V) is present in ionic forms (H₂AsO₄⁻ and HAsO₄²⁻), which are strongly adsorbed onto various soil minerals, such as iron and aluminum (hydr)oxides (4–6). In contrast, the adsorption of As(III) is less than that of As(V) because it is present in a nonionic form (H₃AsO₃) under neutral pH conditions (4–6). Thus, the development of anoxic conditions in soils and aquifers leads to an increase in arsenic mobility. Globally, elevated levels of arsenic in groundwater are a major threat to public health. Bangladesh and West Bengal are among the regions that are severely affected by arsenic contamination in groundwater. In these regions, approximately 60 to 100 million people are exposed to more than 10 μg · liter⁻¹ of arsenic in drinking water, which is the health standard set by the World Health Organization (3, 7).

Bacteria and archaea can reduce As(V) by a detoxification process or by an energy-conserving respiratory process (8–10). The detoxification process is known as the arsenic resistance system (Ars system), which mainly comprises ArsR (a regulatory protein), ArsC [a cytoplasmic As(V) reductase], Acr3 or ArsB [an As(III) efflux pump], ArsA (a pump-driving ATPase), and ArsD (a chaperone of the pump) (9). However, the Ars system may play a minor role in the release of arsenic that is adsorbed onto the soil minerals, as it can only reduce dissolved As(V) in the liquid phase (11). In contrast, the dissimilatory As(V)-reducing prokaryotes are involved in the release of arsenic from flooded soils and anoxic sediments (12, 13). Such prokaryotes can utilize As(V) as a terminal electron acceptor for anaerobic growth through a dissimilatory As(V) reductase designated Arr. Arr, which belongs to the dimethyl sulfoxide (DMSO) reductase family, is a heterodimeric or heterotrimeric molybdoprotein (14, 15). ArrA is a large catalytic subunit that contains a [4Fe-4S] cluster and molybdopterin as a cofactor. ArrB is a small electron transfer subunit with three to four [4Fe-4S] clusters. In some cases, a membrane anchor subunit (ArrC) is present. Currently, the Arr of Chrysiogenes arsenatis (16), Shewanella sp. ANA-3 (17, 18), and Bacillus selenitireducens (19), which are localized to the cytoplasmic membrane or periplasmic space, have been well characterized.

Previously, a novel As(V)-reducing bacterium, designated strain PSR-1, was isolated from arsenic-contaminated soil in Japan (20). The strain was closely related to Anaeromyxobacter dehalogenans, with 16S rRNA gene sequence similarity of 99.7%. The strain PSR-1 can release arsenic from As(V)-adsorbed ferrihydrite or from arsenic-contaminated soil. A. dehalogenans, a facultative anaerobic myxobacterium within the class Deltaproteobacteria, can utilize various electron acceptors, such as halogenated phenols, nitrate, nitric oxide, fumarate, oxygen, Fe(III), U(VI), and Se(IV) (21–25). Recent studies have demonstrated that Anaeromyxobacter spp. are involved in arsenic release from soils and sediments (26–31). However, strain PSR-1 is the only cultured Anaeromyxobacter bacterium known to respire As(V). Interestingly, the genes encoding Arr (arrAB) were not identified in the draft genome of strain PSR-1, although three distinct ars gene clusters were found (32). In addition, attempts to amplify a putative arrA gene by using multiple PCR primers were unsuccessful (20). These results suggest that As(V) reduction by strain PSR-1 is catalyzed by hitherto unknown proteins. Previously, it was suggested that tetrathionate reductase or ArsC plays a role in dissimilatory As(V) reduction by certain prokaryotes, which lack the typical arrAB genes (33, 34).

In this study, we aimed to identify the dissimilatory As(V) reductase of strain PSR-1 by a determination of peptide sequences obtained from an active protein band of in-gel enzyme assay developed for As(V)-reducing activity. In addition, the transcription of genes potentially involved in As(V) respiration was quantified. To identify the proteins that are specifically or abundantly expressed under As(V)-respiring conditions, comparative proteomic analysis using the extracts of the cells grown on As(V) or fumarate as the electron acceptor was also performed by a combination of one-dimension SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

RESULTS

Activity and localization of dissimilatory As(V) reductase.

The dissimilatory As(V) reductase activity in the cell extracts of strain PSR-1, which was grown on 20 mM As(V) or fumarate, was determined using reduced methyl viologen as an electron donor (see Table S1 in the supplemental material). Dissimilatory As(V) reductase activity under As(V)-respiring conditions (35.7 U · mg protein⁻¹) was 16-fold higher than that under fumarate-respiring conditions (2.19 U · mg protein⁻¹). To determine localization of the dissimilatory As(V) reductase, the periplasmic fraction was prepared from washed whole cells by lysozyme-EDTA treatment. As shown in Table S2 in the supplemental material, 89% of the total activity was found in the periplasmic fraction. The activity of malate dehydrogenase, a cytoplasmic marker enzyme, was detected predominantly (92%) in the spheroplast fraction, indicating that the periplasmic fraction was prepared appropriately.

Activity staining and LC-MS/MS analysis.

After the periplasmic fraction was run on an SDS-PAGE gel under mild denaturing conditions, an in-gel enzyme assay was developed for As(V)-reducing activity. A single clear band appeared on the gel (Fig. 1A), and this As(V)-reducing band was excised and run out on a fully denaturing SDS-PAGE gel. As shown in Fig. 1B, a band (band A) with an apparent molecular weight of 116 kDa was observed after Coomassie brilliant blue (CBB) staining. A thick band (band B) with the same molecular weight (116 kDa) was obtained upon directly subjecting the periplasmic fraction to SDS-PAGE under fully denaturing conditions (Fig. 1B). These two bands were excised, digested with trypsin, and subjected to LC-MS/MS analysis. Band A comprised 96 proteins with apparent molecular weights ranging from 7 to 159 kDa (see Table S3A in the supplemental material). Of these proteins, a protein with a locus tag of PSR1_00330, annotated as a catalytic subunit of molybdopterin oxidoreductase, exhibited the highest sequence coverage at 76% (Table 1). Band B comprised 39 proteins (Table S3B), and the same protein (PSR1_00330) exhibited the highest sequence coverage at 79% (Table 1). From band B, another molybdopterin oxidoreductase (PSR1_00324) and formate dehydrogenase-N subunit alpha were also detected with high sequence coverages of 43% and 64%, respectively. The latter protein was also detected in band A with sequence coverage of 40% (Table S3A). Formate dehydrogenase-N subunit alpha protein is also known as a member of the molybdopterin oxidoreductases (35).

FIG 1.

(A) Activity staining of dissimilatory arsenate reductase of Anaeromyxobacter sp. PSR-1. The concentrated periplasmic fraction was denatured partially (2% sodium dodecyl sulfate [SDS] on ice for 5 min). The fraction was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 4°C. The gel was stained anaerobically with methyl viologen and arsenate as the electron donor and acceptor, respectively. A clear band represents the active band. (B) The excised active band (lane 2) and total periplasmic proteins (lane 3) resolved under completely denatured condition (100°C, 5 min with SDS and 2-mercaptoethanol). The gel was stained with Coomassie brilliant blue R-250. Lane 1 represents the standard marker proteins, although many faint bands also appear. The bands with a molecular weight of approximately 116 kDa (bands A and B) were excised, digested with trypsin, and subjected to liquid chromatography-tandem mass spectrometry analysis (see Table 1). Note that the images of lanes 1, 2, and 3 were obtained from one gel.

TABLE 1.

Top 5 proteins identified from bands A and B in LC-MS/MS analysis^a

Band and protein name	Locus tag	Predicted mol wt (kDa)	No. of exclusive unique peptide counts	Sequence coverage (%)
Band A
Molybdopterin-dependent oxidoreductase	PSR1_00330	118	72	76
Bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase	PSR1_00860	91	40	58
Malate dehydrogenase	PSR1_01530	33	12	54
Metal-dependent hydrolase	PSR1_02748	28	8	52
DUF3857 domain-containing protein, partial	PSR1_04203	136	47	48
Band B
Molybdopterin-dependent oxidoreductase	PSR1_00330	118	77	79
Formate dehydrogenase-N subunit alpha	PSR1_02929	112	45	64
Molybdopterin-dependent oxidoreductase	PSR1_00324	119	42	43
Isoleucine-tRNA ligase	PSR1_00494	106	35	39
Acetyl-CoA hydrolase/transferase family protein	PSR1_01498	56	11	31

^aAll proteins are listed in descending order of the sequence coverage (%).

Comparative proteomic analysis of strain PSR-1 grown on As(V) and fumarate.

In the comparative proteomic analysis, a total of 1,782 proteins were identified, and these corresponded to 40% of the predicted protein-coding sequence (CDS) of strain PSR-1. Of these proteins, 1,302 proteins were detected in both As(V)- and fumarate-grown cells, and these are summarized in Table S4 in the supplemental material. In contrast, specific induction of 163 and 317 proteins was observed in As(V)- and fumarate-grown cells, respectively (see Tables S5 and S6 in the supplemental material).

As shown in Table 2, the protein PSR1_00330 identified in LC-MS/MS analysis was among the top 5 proteins, which were more highly abundant in As(V)-respiring cells than in fumarate-respiring cells. The top 5 proteins also included a universal stress protein (UspA) and two arsenical pump-driving ATPases (ArsA). In addition, as shown in Table 3, an electron transfer subunit of molybdopterin oxidoreductase (PSR1_00329) was among the 5 most abundant proteins whose expression was observed only in As(V)-respiring cells and not in fumarate-respiring cells. These 5 most abundant proteins also included Acr3, an As(III) efflux transporter.

TABLE 2.

Top 5 proteins whose expression was much more abundant in arsenate-respiring cells than in fumarate-respiring cells^a

Protein name	Locus tag	Relative protein content (%) in cells grown with:		Fold change
		Arsenate	Fumarate
Universal stress protein	PSR1_00119	2.3	0.043	54
Arsenical pump-driving ATPase	PSR1_00127	0.088	0.0031	28
Arsenical pump-driving ATPase	PSR1_00118	2.6	0.093	28
Molybdopterin oxidoreductase	PSR1_00330	0.058	0.0022	26
Acetamidase	PSR1_00977	0.050	0.0020	25

^aAll proteins are listed in descending order of the fold change (arsenate/fumarate) of expression.

TABLE 3.

Top 5 proteins whose expression was only observed in arsenate-respiring cells

Protein namea	Locus tag	Relative protein content (%)
3-Oxoacyl-ACP reductase FabG	PSR1_04088	0.061
4Fe-4S dicluster domain-containing protein	PSR1_00329	0.035
Hypothetical protein	PSR1_00111	0.027
ACR3 family arsenite efflux transporter	PSR1_00121	0.026
Hypothetical protein	PSR1_00091	0.025

^aProteins are listed in descending order of the relative protein content (%).

Structural motif prediction and phylogenetic analysis of molybdopterin oxidoreductases.

The genes encoding the catalytic (PSR1_00330) and electron transfer (PSR1_00329) subunits of molybdopterin oxidoreductase were predicted to form an operon-like structure on the genome of strain PSR-1 (gene locus identifier [ID] of psr1_00330 and psr1_00329; Fig. 2). The protein PSR1_00330 was a typical member of the large catalytic subunit of the DMSO reductase family, which is reported to contain a molybdo-bis (pyranopterin guanine dinucleotide) cofactor at the active site (36–38). As shown in Fig. S1 in the supplemental material, the protein PSR1_00330 included a twin-arginine translocation (TAT) signal sequence (RREFL) at the N-terminal region, a motif associated with a [4Fe-4S] cluster (CX₂CX₃CX_24–26H) (36), and a molybdopterin guanine dinucleotide coordination motif. Phylogenetic analysis revealed that the protein PSR1_00330 belongs to the tetrathionate reductase (TtrA) clade in the DMSO reductase family (Fig. 3). In contrast, the protein PSR1_00329 harbored four [4Fe-4S]-binding motifs (CX₂CX_2–11CX₃C), which is a typical characteristic of the electron transfer subunit of tetrathionate reductase (TtrB) (Fig. S1). Phylogenetic analysis of the protein PSR1_00329 suggested that TtrB-related proteins did not form a single clade but consisted of three distinct clades within the small electron transfer subunits of the DMSO reductase family (see Fig. S2 in the supplemental material). Although the putative psr1_00329-psr1_00330 operon lacked a gene encoding a membrane anchor subunit of tetrathionate reductase (TtrC), a gene encoding a c-type cytochrome (cyt c) was adjacent to these two genes (Fig. 2). Interestingly, another putative ttr-like operon (psr1_00323-psr1_00324) was observed upstream of the psr1_00329 and psr1_00330 genes (Fig. 2), and the protein PSR1_00324 was detected in band B (Table 1). The protein PSR1_00324 exhibited a 53% sequence similarity with PSR1_00330 and had similar motifs and TAT signal sequence (RRDFL) to those of the protein PSR1_00330 (Fig. S1). Similarly, the protein PSR1_00323, which exhibited 52% sequence similarity with PSR1_00329, harbored four [4Fe-4S]-binding motifs (Fig. S1). Of the 3 ars operons, one operon was observed in the vicinity of the psr1_00323 and psr1_00324 genes (Fig. 2).

FIG 2.

Two distinct gene clusters encoding tetrathionate reductase-related proteins were found in the draft genome (GenBank accession number NZ_BAZG00000000.1) of Anaeromyxobacter sp. PSR-1. Tetrathionate reductase gene homologs, a c-type cytochrome gene, and putative ars genes are represented in orange, green, and cyan, respectively. Other genes are represented in gray. HK, histidine kinase; HP, hypothetical protein; RR, response regulator.

FIG 3.

Phylogenetic analysis of the proteins PSR1_00330 and PSR1_00324 relative to other oxidoreductases within the dimethyl sulfoxide (DMSO) reductase family. Neighbor-joining phylogenetic analysis was performed using MEGA7. Bar, 0.2 amino acid changes per site. AioA, arsenite oxidase; ArrA, arsenate reductase; ArxA, anaerobic arsenite oxidase; BisC, biotin-d-sulfoxide reductase; ClrA, chlorate reductase; DorA, DMSO reductase; Fdh, formate dehydrogenase; NapA, periplasmic nitrate reductase; PhsA, thiosulfate reductase; PsrA, polysulfide reductase; SerA, selenate reductase; TorA, TMAO reductase; TtrA, tetrathionate reductase.

Transcriptional analysis of genes potentially involved in As(V) reduction.

To test whether psr1_00329 and psr1_00330 genes are cotranscribed as a single operon, the primers for reverse transcription-PCR (RT-PCR) were designed to span the following regions: phnD and cyt c (RT1), cyt c and psr1_00329 (RT2), and psr1_00329 and psr1_00330 (RT3) (see Table S7 in the supplemental material). Total RNA was extracted from the cells grown on As(V), and cDNA was synthesized from the extracted RNA. PCR products were observed in primer pairs RT2 and RT3 but not in RT1 (see Fig. S3 in the supplemental material). These results suggest that cyt c, psr1_00329, and psr1_00330 are transcribed together in a single mRNA, whereas phnD and cyt c are not cotranscribed.

The expression levels of psr1_00330 and psr1_00324 genes relative to the expression of the recA gene were quantified and compared using the RNA extracted from the cells grown on As(V) or fumarate. In addition, a gene encoding formate dehydrogenase-N subunit alpha (fdnG) was also quantified, as this molybdopterin oxidoreductase was detected in both bands A and B with high sequence coverages (Table 1). As shown in Table 4, the expression of psr1_00330 in the cells grown on As(V) was 19-fold higher than that in the cells grown on fumarate (1.3 versus 0.069). However, the expression levels of the psr1_00324 gene were similar among the cells grown on As(V) and fumarate (1.6 versus 1.9). In contrast, the expression of fdnG in the cells grown on As(V) was lower than that in the cells grown on fumarate (1.4 versus 13).

TABLE 4.

Expression of psr1_00330, psr1_00324, and fdnG genes relative to that of the recA gene in arsenate- and fumarate-grown cells

Gene	Mean ± SD under growth with:a
	Arsenate	Fumarate
psr1_00330	1.3 ± 0.39	0.069 ± 0.0053
psr1_00324	1.6 ± 0.22	1.9 ± 0.28
fdnG	1.4 ± 0.48	13 ± 2.0

^aValues represent the ratio of the relative quantity of psr1_00330, psr1_00324, or fdnG transcripts to that of recA gene transcripts. Data are means ± standard deviations (n = 3).

Growth on tetrathionate and tetrathionate reductase activity.

Tetrathionate reductase is reported to catalyze tetrathionate (S₄O₆²⁻) reduction to form thiosulfate (S₂O₃^2–) in Salmonella enterica. Tetrathionate reductase enables S. enterica to utilize tetrathionate as the terminal electron acceptor for anaerobic growth (39, 40). To evaluate the ability of dissimilatory As(V) reductase of strain PSR-1 to reduce tetrathionate, the strain was grown in the basal medium containing tetrathionate as the electron acceptor. However, strain PSR-1 did not exhibit growth on tetrathionate (see Fig. S4A in the supplemental material). Furthermore, crude extracts of strain PSR-1 grown on As(V) or fumarate did not exhibit significant tetrathionate reductase activity (Table S1). Finally, the ability of tetrathionate reductase of S. enterica to catalyze As(V) reduction was determined. Although S. enterica JCM1651 could grow on tetrathionate under anaerobic conditions (Fig. S4B), it did not exhibit growth on As(V) as the electron acceptor (data not shown). The crude extracts of strain JCM1651 grown on tetrathionate exhibited 26.0 U · mg protein⁻¹ of tetrathionate reductase activity (see Table S8 in the supplemental material). However, the crude extract did not exhibit significant As(V) reductase activity. The crude extracts of strain JCM1651 cultured in Luria Bertani (LB) medium under aerobic conditions or on glucose under anaerobic conditions did not exhibit tetrathionate reductase or As(V) reductase activity (Table S8).

DISCUSSION

The dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions, which concurred with the results of previous studies on Chrysiogenes arsenatis and Geobacter sp. OR-1 (16, 41). In addition, the protein was localized in the periplasmic space, which was consistent with the results of previous studies on the Arr of Shewanella sp. ANA-3 and Geobacter sp. OR-1 (18, 41). However, the multiple proteomic analyses of this study suggested that the protein was not a classical Arr and that the protein was closely related to the tetrathionate reductase of S. enterica. The protein PSR1_00330, which is closely related to TtrA, exhibited the highest sequence coverage in the LC-MS/MS analysis among the proteins recovered from the active band (Table 1, band A). Additionally, the expression of PSR1_00330 was found to be abundant in the periplasmic fraction (Table 1, band B). Furthermore, comparative proteomic analysis revealed that both PSR1_00330 and PSR1_00329 were among the five most abundant proteins that were differentially and specifically expressed, respectively, in the As(V)-respiring cells (Tables 2 and 3). The transcription of the psr1_00330 gene was upregulated in the As(V)-respiring cells of strain PSR-1 (Table 4). Based on these results, it is proposed that the protein PSR1_00330 functions as a respiratory As(V) reductase in strain PSR-1. In this study, we demonstrated the involvement of a TtrA homolog in dissimilatory As(V) reduction both at the mRNA and protein levels. However, further biochemical or genetic determination will be required to fully characterize the physiological role of the protein PSR1_00330.

The potential involvement of TtrA homologs in As(V) respiration was first reported in several archaeal species (33). Pyrobaculum aerophilum, an As(V)-respiring archaeon, has no typical arrA gene in its genome. However, a gene encoding a putative molybdopterin oxidoreductase (PAE1265) was highly upregulated under As(V)-respiring conditions. The PAE1265 protein exhibited a 27% sequence similarity with TtrA of S. enterica. A gene orthologous to PAE1265, Pars_0389, was detected in Pyrobaculum arsenaticum, the only other known As(V)-respiring archaeon. In Pyrobaculum calidifontis, which does not exhibit As(V) respiration, an orthologue of PAE1265 (Pcal_1601) was truncated after 810 amino acids by a stop codon (TAG). Interestingly, a persistent effort to grow P. calidifontis on As(V) enabled this archaeon to exhibit As(V) respiration. The genome resequence analysis of the As(V)-respiring P. calidifontis revealed that the TAG stop codon in Pcal_1601 was replaced with a GAG Glu codon (33). These results suggest that As(V) respiration by Pyrobaculum spp. is catalyzed by the proteins homologous to TtrA.

Similar observations have been reported in several As(V)-respiring bacteria. Melioribacter roseus, a member of the phylum Chlorobi, exhibits As(V) respiration but does not have the typical arrA gene (42). This bacterium harbors Mros_1076 and Mros_1774; these genes each encode a form of putative molybdopterin oxidoreductase, sharing homology with TtrA and thiosulfate/polysulfide reductase (PsrA/PhsA), respectively. The transcription of both genes was reported to be markedly upregulated under As(V)-respiring conditions (43). A gene orthologous to Mros_1076 was also found in Ignavibacterium album, another As(V)-respiring Chlorobi bacterium, which does not contain the typical arrA gene (42). Recently, Tsuchiya et al. (41) identified dissimilatory As(V) reductase of Geobacter sp. OR-1 by LC-MS/MS analysis of proteins recovered from the active band, which was the same strategy used in this study. In addition to the typical ArrA, another protein closely related to TtrA was also detected with high sequence coverage. The study also reported that the expression of the TtrA homolog and its corresponding gene were upregulated under As(V)-respiring conditions (41). As shown in Fig. 3, the TtrA homologs in M. roseus, I. album, and Geobacter sp. OR-1 are phylogenetically closely related to the protein PSR1_00330 of strain PSR-1. These results suggest that the proteins homologous to TtrA may be involved in As(V) respiration not only in archaea but also in bacteria.

Although the proteins PSR1_00330 and PSR1_00329 shared significant homologies with TtrA and TtrB of S. enterica, they exhibit several distinctive features. The putative TAT signal sequence was observed only in the catalytic large subunit of PSR1_00330 (Fig. S1), whereas this motif was observed in both TtrA and TtrB of S. enterica. In addition, no gene orthologous to that encoding the membrane anchor subunit TtrC was found in the strain PSR-1. The psr1_00329 and psr1_00330 genes were adjacent to the gene encoding a c-type cytochrome. As this gene was cotranscribed with the genes psr1_00329 and psr1_00330 (Fig. S3), it might also be involved in As(V) respiration as an electron carrier. Furthermore, putative genes encoding TtrS and TtrR, which are the sensor kinase and response regulator of a two-component transcriptional regulatory system, respectively (39, 40), were not observed in the vicinity of the psr1_00330 gene. Further studies are needed to understand the transcriptional regulation of the psr1_00330 gene by arsenic.

In S. enterica, the ttrB, ttrC, and ttrA genes form an operon (39, 40). As shown in Fig. S5 in the supplemental material, the putative ttrBCA operon is distributed widely in the TtrA clade of the DMSO reductase family, including Enterobacteriaceae, archaea, and various bacteria, such as M. roseus and I. album. In contrast, the distribution of a putative cytc-ttrBA operon is limited to Deltaproteobacteria, such as Geobacter sp. OR-1 and strain PSR-1. Thus, it is difficult to predict the function of genes related to ttrA from their operon organization. The results of this study also suggest that the membrane anchor subunit is not always necessary for As(V) reduction by the proteins homologous to TtrA.

In dissimilatory As(V)-reducing bacteria, such as Shewanella sp. ANA-3 and Geobacter sp. OR-1, the arrAB genes are flanked by the ars genes (17, 41). Similarly, the putative ars genes were present in the vicinity of the psr1_00329 and psr1_00330 genes in strain PSR-1 (Fig. 2). These ars genes may play important roles by conferring arsenic resistance to the cells of strain PSR-1 by reducing the As(V) entering the cells to As(III), which is pumped out of the cells via Acr3. Two ArsA proteins and an Acr3 protein were among the five most abundant proteins in the As(V)-respiring cells, which suggested their importance during As(V) respiration. Generally, Anaeromyxobacter spp. have 5 to 7 ars genes, whereas strain PSR-1 harbors at least 18 putative ars genes in its genome (see Table S9 in the supplemental material). This might reflect the high arsenic content (1,000 mg · kg⁻¹) of the original soil environment from which strain PSR-1 was isolated (20).

The redox potentials of the As(V)/As(III) couple and the tetrathionate/thiosulfate couple are very close (between −10 and +80 mV at pH 7.0). This may imply that TtrA and related proteins can recognize both As(V) and tetrathionate as substrates due to their similar redox potentials. However, the results of this study suggest that TtrA of S. enterica cannot reduce As(V) and that the PSR1_00330 protein cannot reduce tetrathionate (see Fig. S4, Table S1, and Table S8 in the supplemental material). The TtrA clade of the DMSO reductase family also includes selenate (SeO₄²⁻) reductase (SrdA) of Bacillus selenatarsenatis (44) (Fig. 3). Hence, this clade may comprise multiple proteins with different substrate specificities, which are observed in the PsrA/PhsA clade comprising polysulfide reductase, thiosulfate reductase, and selenite reductase (45). Although the extent of distribution of the TtrA-related dissimilatory As(V) reductases among prokaryotes is still unclear, it would not be surprising if such proteins play an important role along with ArrA in the reduction and subsequent release of arsenic from soils and sediments. Furthermore, the findings of this study demonstrate the role of the TtrA clade, which is a diverse group in the DMSO reductase family, in the biogeochemical cycling of toxic metalloids.

MATERIALS AND METHODS

Growth conditions and culture media.

Strain PSR-1 was previously isolated from the arsenic-contaminated soil in Japan (20). The strain was routinely cultured under anaerobic conditions at 30°C in a basal medium. The composition of the culture medium was as follows (for 1 liter): 0.54 g NH₄Cl, 0.14 g KH₂PO₄, 0.20 g MgCl₂·6H₂O, 0.15 g CaCl₂·2H₂O, 0.14 g Na₂SO₄, 2.5 g NaHCO₃, 1.0 ml vitamin solution, 1.0 ml trace metal solution, and 1.0 ml selenite/tungstate solution. The medium was sterilized by autoclaving at 121°C for 20 min. Acetate (10 mM), As(V) (20 mM as KH₂AsO₄), and cysteine-HCl (1 mM) were added to the medium from sterile anaerobic stock solutions as the electron donor, electron acceptor, and reducing agent, respectively. The medium (20 ml) was dispensed into a 60-ml serum bottle under an N₂-CO₂ (80:20 [vol/vol]) gas stream. The bottle was sealed with a thick butyl rubber stopper and an aluminum cap. In some cases, fumarate (20 mM) or tetrathionate (20 mM) was used as the electron acceptor.

Salmonella enterica subsp. enterica JCM1651 was routinely cultured under aerobic conditions at 30°C on LB agar medium. The JCM1651 cells cultured in the LB medium under aerobic conditions were inoculated into the basal medium containing acetate (10 mM) and tetrathionate (20 mM). In some cases, As(V) was added to the medium instead of tetrathionate. When strain JCM1651 was grown fermentatively, cells cultured under anaerobic conditions were inoculated into the basal medium containing 20 mM glucose as a substrate.

Preparation and fractionation of crude cell extracts.

The crude cell extracts were prepared using the cells grown on As(V), fumarate, or tetrathionate. The cells were washed twice and resuspended in 20 mM Tris-HCl buffer (pH 6.8). The cells were disrupted by sonication (Q500 sonicator; Qsonica, Newtown, CT) at 20 kHz and 30% amplitude for 3 min. The cell lysate was centrifuged at 17,000 × g and 4°C for 30 min to remove the cell debris. The periplasmic fraction was prepared from the washed whole cells as described previously (46). Briefly, the cells were incubated in a buffer containing 20 mM Tris-HCl buffer (pH 6.8), 1 mM Na₂-EDTA, 0.5 M sucrose, and 25 mg · ml⁻¹ lysozyme at 30°C for 20 min. The periplasmic fraction was separated from the spheroplasts and nonlysed cells by centrifugation at 17,000 × g and 4°C for 30 min. The disruption of spheroplasts and removal of cell debris were performed as described previously (46).

Enzyme assays.

The reductase activities were assayed spectrophotometrically in a sealed quartz cuvette at 30°C by monitoring the oxidation of reduced methyl viologen (ε₅₇₈ = 9.7 mM⁻¹ · cm⁻¹) as an electron donor. The reaction mixture (0.7 ml) containing 20 mM Tris-HCl (pH 6.8), 0.3 mM methyl viologen, an appropriate amount of enzyme, and 10 mM As(V) or 0.36 mM tetrathionate was degassed and sparged with N₂ gas. The reaction was initiated with the addition of sodium dithionite to obtain an absorbance of 1.5 to 2.0. One unit (U) of the reductase activity was defined as the amount of enzyme protein required to oxidize 1 μmol of reduced methyl viologen per minute. The protein concentration was determined using the protein assay kit (Bio-Rad, Hercules, CA). Bovine serum albumin was used as a standard protein. Malate dehydrogenase activity was measured spectrophotometrically by monitoring the rate of disappearance of NADH (47). The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.2 mM NADH (ε₃₄₀ = 6.22 mM⁻¹ · cm⁻¹), 0.2 mM oxaloacetate, and an appropriate amount of enzyme.

Electrophoresis, activity staining, and LC-MS/MS analyses.

To resolve the proteins of the periplasmic fraction, electrophoresis was performed in two steps. In the first step, partially denatured samples (2% SDS) were subjected to electrophoresis at 4°C using 8% polyacrylamide gel in 25 mM Tris-glycine buffer (pH 8.3), following the method described by Laemmli (48). Next, the gel was incubated under a nitrogen atmosphere with 20 mM Tris-HCl (pH 6.8) containing 0.4 mM methyl viologen, 12.5 mM As(V), and 6 mM dithionite. In the second step, the proteins from the clear band (active band), which was observed on the gel, were excised, boiled for complete denaturation (with 2% SDS and 5% 2-mercaptoethanol for 5 min), and then subjected to SDS-PAGE. Protein molecular weight marker (TaKaRa, Otsu, Japan) was used as the standard marker protein. The proteins were visualized by staining the gel with CBB R-250. The target band was excised, digested with trypsin, and subjected to LC-MS/MS analysis as described previously (49).

Transcriptional analysis.

RNA was prepared from triplicate cultures of cells grown on As(V) or fumarate. Cells at the mid-exponential phase of growth were collected by centrifugation at 10,000 × g and 4°C for 10 min. The cell pellet was immediately frozen in a liquid nitrogen bath and stored at −80°C. Total RNA was extracted using the RNeasy miniprep kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The eluent was then processed to remove the residual DNA using the Turbo DNA-free kit (Ambion, Carlsbad, CA). To ensure that the RNA samples were not contaminated with genomic DNA, the samples were subjected to PCR amplification with primers targeting the 16S rRNA gene (338F and 518R) (50). The DNase-treated RNA samples were stored at −80°C until use. All RNA samples had A₂₆₀/A₂₈₀ ratios of 1.8 to 2.0, which indicated high purity.

The cDNA was synthesized using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The RNA samples were reverse transcribed using random hexamers. A control containing no SuperScript IV reverse transcriptase (Invitrogen) was included for each RNA sample to ensure that DNA contamination did not affect mRNA detection. The RNA extraction and subsequent cDNA synthesis were performed in triplicates. Thus, three separate cDNA samples were obtained, which were subjected to RT-PCR.

The genes potentially involved in As(V) reduction were mapped using the total RNA extracted from cells grown on As(V). The cDNA was synthesized as described above. The cDNA was subjected to PCR using various combinations of primers (see Table S7 in the supplemental material) to determine whether the target genes are cotranscribed as a single operon. The primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). The PCR conditions were as follows: 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and 72°C for 10 min.

RT-PCR.

The expression of psr_00330, psr1_00324, and fdnG genes was analyzed using the cDNA samples by RT-PCR. The expression levels of target genes were normalized to those of housekeeping gene recA. The gene expression levels were presented as the number of target gene transcripts per recA gene transcript. The new primers designed for RT-PCR are shown in Table S10 in the supplemental material. The amplicon sizes were limited to <150 bp for accurate quantification. The RT-PCR analysis was performed using SYBR green in the Step One Plus instrument (Applied Biosystems). The RT-PCR analysis was performed in a 20-μk reaction mixture containing 10 μl of Power SYBR green PCR mastermix (Applied Biosystems), 0.4 μl (5 μM concentration) of each primer, 2 μl diluted template (cDNA), and 7.2 μl of nuclease-free water. The PCR conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. The analysis was performed in triplicates. There was no amplification of the no-template controls and DNase-treated RNA samples. A final dissociation step was performed to obtain the melting curves (thermal profile) of the amplicons. The standard curves were generated using serially diluted (covering a range of 5 orders of magnitude) cDNA samples prepared from the cells grown on As(V). The standard curves were generated by plotting the threshold cycle (C_T) values as a function of the log of the amount of cDNA. The slopes of standard curves were used for calculation of the PCR efficiencies as follows:

$$E\hspace{0.17em}(\%)=[{10}^{(-1/\text{slope})}-1]\times 100$$

The E values obtained for all genes and primer pairs ranged from 95% to 103%, and R² values ranged from 0.999 to 1.000 (Table S10).

Proteomic analysis.

The cells grown on As(V) or fumarate as the electron acceptor were harvested at the early stationary phase by centrifugation at 6,000 × g and 4°C for 15 min. The cells were washed twice with 8% NaCl. The harvested cells were lysed using the ReadyPrep protein extraction kit (Bio-Rad, Hercules, CA). Equal amounts of total proteins (50 μg) were subjected to SDS-PAGE using 10% gel (90 mm × 85 mm). The resolved proteins were stained with CBB R-250. The gel lanes were cut into 45 1 mm-long strips. The gel strips were completely destained with 30% acetonitrile (ACN) in 25 mM NH₄HCO₃, reduced with 10 mM dithiothreitol, and alkylated with 55 mM iodoacetamide. After these gel strips dried completely, in-gel digestion was performed using 40 μl of sequencing-grade modified trypsin (12.5 μg · ml⁻¹ in 50 mM NH₄HCO₃) at 37°C overnight. The digested peptides were extracted with 25 mM NH₄HCO₃ in 60% ACN and further extracted twice with 5% formic acid in 70% ACN.

The LC-MS/MS analysis was performed using an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with a multidimensional high-performance liquid chromatography (HPLC) Paradigm MS2 (AMR, Tokyo, Japan) and a nanospray electrospray ionization device (Michrom Bioresources, Auburn, CA) as described previously. The peptide spectra were recorded in a mass range of m/z 450 to 1,800. The obtained MS/MS data were searched against the data on strain PSR-1 in the National Center for Biotechnology Information (NCBI) database (GenBank accession number NZ_BAZG00000000.1) using the Mascot program v2.5 (Matrix Science, London, UK). The protein abundance index (PAI) was defined as follows: PAI = N_observed/N_observable, where N_observed and N_observable are the numbers of experimentally observed peptides and the number of theoretically observable peptides for each protein, respectively (51–53). The label-free quantitative analysis of the abundance of the identified proteins was performed using the exponentially modified PAI (emPAI) values that are provided by the Mascot program, as follows:

$${\text{emPAI = 10}}^{\text{PAI}}-\text{1}$$

The relative protein content in molar fraction percentage was then determined using the following equation:

$$\text{relative protein content}(\text{mol \%})\text{= emPAI/Σ}(\text{emPAI})\times 100$$

where Σ(emPAI) is the summation of the emPAI values for all of the identified proteins (51, 52).