Identification of Multiple Soluble Fe(III) Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III) and Cr(VI) anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI) reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III) reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III) reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS) with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs), thioredoxin reductase (Trx), NADP(H)-nitrite reductase (Ntr), and thioredoxin disulfide reductase (Tdr) were determined to be responsible for Fe(III) reductase activity. Amino acid sequence and three-dimensional (3D) structural similarity analyses of the T. indiensis Fe(III) reductases were carried out with Cr(VI) reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI) reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI) reduction as well.


Introduction
The involvement of iron in a number of physiological functions, such as a cofactor for enzymes or an electron acceptor in anaerobic respiration, of most organisms has been recognized [1]. However, most of the metabolic functions of iron require the Fe(II) oxidation state which is more soluble and, therefore, more bioavailable than the Fe(III) form [2].
One of the strategies bacteria have evolved for assimilation and utilization of iron is the reduction of Fe(III) to Fe(II) [3]. Bacterial iron reduction has been studied extensively and various biomolecules responsible for Fe(III) reduction have been discovered [4]. Fe(III) reductases involved in anaerobic respiration are either secreted into the extracellular medium or localized in the cell membrane [5]. Nonetheless, most of the known Fe(III) reductases have been found to be soluble, residing in either the cytoplasm or the periplasmic space [5]. Further, based on the fact that Fe(III) is solubilized by chelators it has been suggested that even soluble Fe(III) reductase could participate in anaerobic respiration [6].
We have earlier reported that a low-GC content Gram-positive Thermoanaerobacter-like bacterium (now designated as Thermoanaerobacter indiensis BSB-33) reduces Fe(III) and Cr(VI) anaerobically at 60 ∘ C optimally [8]. The sequence of T. indiensis genome has been determined and annotated (http://www.jgi.doe.gov). Taking advantage of this we have attempted to identify the soluble proteins which are involved in Fe(III) reduction by a combination of chromatography and mass spectrometry (MS). Bioinformatic analysis has also been carried out to find sequence and threedimensional structural similarity of these proteins with known Cr(VI) reductases.

Materials and Methods
2.1. Strain and Culture. T. indiensis BSB-33 (ATCC BAA-2171) cells were grown anaerobically in Luria-Bertani (LB) medium at 60 ∘ C in Hungate bottles under 100% N 2 atmosphere. Transfers and sampling of cultures were carried out anaerobically.

Enzyme
Assays. Fe(III) reduction assay was performed by a modification of the procedure of Kaufmann and Lovley [6] under anaerobic conditions at 60 ∘ C in a mixture containing 50 mM HEPES (pH 7.0), 1 mM Fe(III)-citrate, 1 mM NADH, 1 mM ferrozine, and 30 to 100 g of protein.
One unit (U) is defined as the amount of enzyme reducing 1 mol of Fe(III) per min. Cr (VI) reduction assay was carried out anaerobically at 60 ∘ C as described earlier [9]. In-gel Fe(III) reduction assay was done by 10% nondenaturing polyacrylamide gel electrophoresis (PAGE) of protein fractions. The gel was stained according to the protocol of Gaspard et al. [10].

Cell Fractionation.
Cells were harvested by centrifugation at 400 g for 10 min and washed twice in N 2 -saturated Tris-HCl buffer (pH 7.0) containing 75 mM NaCl, 75 mM KCl, 20 mM MgCl 2 , and 0.1 mM dithiothreitol. 10 g of wet cells was resuspended in 70 mL of the same buffer, 200 L DNase I (20 mg/mL) and 40 L RNase I (20 mg/mL) were added, and the suspension was stirred on ice for 1 h. Cells were disrupted by three passages through a French pressure cell at 8,000 psi. The crude lysate was centrifuged at 10,000 ×g for 30 min at 4 ∘ C and the supernatant was removed and centrifuged at 100,000 ×g for 1 h at 4 ∘ C. The supernatant was used as the soluble fraction whereas the pellet was resuspended in 50 mM Tris-HCl, pH 7.0, to be treated as the membrane fraction. Protein was quantified using Bradford reagent.

Chromatography.
The chromatographic procedures followed were modifications of the methods described by Kaufmann and Lovley [6]. The soluble fraction was concentrated by ammonium sulfate precipitation (30 to 80%) and applied to a Superdex 200 prep grade column (1.6 × 60 cm; Amersham Pharmacia Biotech) equilibrated with N 2saturated 50 mM Tris-HCl (pH 7.0) containing 75 mM NaCl, 75 mM KCl, 20 mM MgCl 2 , and 2 mM CaCl 2 . Proteins were eluted with the same buffer. The fractions containing Fe(III) reductase activity were pooled, diluted fourfold, and applied to a HiTrap Q HP Sepharose column (1.6 × 2.5 cm; GE Healthcare Life Sciences) equilibrated with 20 mM Tris-HCL (pH 7.5). Bound proteins were eluted with a linear gradient of 0-0.6 M NaCl. The fractions containing maximum Fe(III) reductase activity were pooled, dialyzed in 10 mM sodium phosphate buffer, pH 6.8, and applied to a HT hydroxyapatite column (1 × 15 cm; BioRad) equilibrated with the same buffer. Bound proteins were eluted with a linear gradient of 10-200 mM sodium phosphate buffer, pH 6.8. Active fractions were dialyzed in 20 mM Tris-HCl, pH 7.0, and applied to a mono Q column (0.5 × 5 cm; GE Healthcare Life Sciences) equilibrated with 20 mM Tris-HCl, pH 7.0, and 0.1 M NaCl. Proteins were eluted using a linear gradient of 0.1-0.5 M NaCl in 20 mM Tris-HCl, pH 7.0.

Localization of Fe(III) Reductase
Activity. Crude extracts of the thermophilic bacterium T. indiensis reduced Fe(III)citrate with NADH as the electron donor. The specific activity of reduction measured at 60 ∘ C was 0.052 mol min −1 mg −1 protein (Table 1). On fractionation both the soluble and membrane fractions were found to contain Fe(III) reductase activity. However, 76% of the NADH-dependent Fe(III) reductase activity in the crude cell extracts was recovered in the soluble fraction ( Table 1). The specific Fe(III) reductase activity in the soluble fraction was also about threefold higher than that in the membrane fraction ( Table 1). The soluble extract resolved by nondenaturing PAGE produced four distinct Fe(III) reductase activity bands 1-4 ( Figure 1).
In order to identify the protein(s) responsible for each of the Fe(III) reductase activities a four-step chromatographic procedure was followed. The activities were separated from each other by passing sequentially through Superdex 200, Q sepharose, hydroxyapatite, and mono Q columns. The gel    filtration step could resolve activities 1 and 2 from activities 3 and 4 ( Figure 1). However, in this step activity 1 could not be resolved completely from activity 2 and, similarly, activity 3 could not be separated from activity 4 ( Figure 1). In the second step gel filtration fraction 7 showing a more intense activity 1 band than activity 2 was passed through a Q Sepharose ion-exchange column. Q Sepharose fraction 8 was found to contain the maximum activity 1 free from activity 2 (Figure 2(a)). Similarly, gel filtration fraction 9 which showed greater activity 2 than activity 1 was loaded on a Q Sepharose column. Fraction 6 from Q Sepharose contained activity 2 free from activity 1 as shown in the zymogram (Figure 2(b)). In a similar manner Q Sepharose chromatography could also resolve activity 3 and activity 4 from gel filtration fraction 12 (Figure 2(c)).
In order to purify each of the four activities from nonferric reductase proteins respective Q Sepharose fractions were then passed through hydroxyapatite and mono Q columns. In each step the number of background protein bands was reduced. However, none of the four activities could be completely purified from nonferric reductase protein background (data not shown). Hence, after the mono Q step the protein bands showing Fe(III) reductase activity in preparative nondenaturing gels were excised and analyzed by MS which has the capability to identify multiple proteins from a mixture [15, http://masspec.scripps.edu/services/proteomics/ dataanal.php].

Dihydrolipoamide
Dehydrogenase. MS spectra obtained from band 1 matched against two dihydrolipoamide dehydrogenases (LPDs) in T. indiensis with high Mascot scores ( Table 2). The two proteins contain 469 (LPD1) and 551 (LPD2) amino acid residues, respectively. 3D structures of LPD1 and LPD2 were predicted by EsyPred3D which used Geobacillus stearothermophilus dihydrolipoamide dehydrogenase crystal structure (PDB ID 1EBD) as the template (Table 3). High sequence similarity and 3D structural identity were found between the two proteins (Table 4). These are likely to be paralogs having similar functions in the cell.
LPDs belong to pyridine nucleotide-disulfide oxidoreductase family and are found in many species as a part of pyruvate dehydrogenase complex [16]. In the bacterium Clostridium kluyveri dihydrolipoyl dehydrogenase (synonymously called dihydrolipoamide dehydrogenase) BfmBC reduces Fe(III) complexes of citrate, ATP, and ADP in an NAD(P)H-dependent manner [2]. It has been proposed that in C. kluyveri NADH transfers a hydride ion to enzymebound flavin and FADH 2 thus produced donates an electron to ferric chelates [2].
NCBI BLAST showed 37% and 39% sequence similarity of C. Kluyveri BfmBC with T. indiensis LPD1 and   LPD2, respectively (Table 4). EsyPred3D also predicted the 3D structure of BfmBC using Geobacillus stearothermophilus dihydrolipoamide dehydrogenase crystal structure (PDB ID 1EBD) as the template (Table 3). Both LPD1 and LPD2 were found to be identical to BfmBC by 3D structural alignment (Table 4). Further, the two T. indiensis proteins as well as C. kluyveri BfmBC contain the most conserved FAD-binding sequence motif XhXhGXGXXGXXXhXXh(X) 8 hXhE(D), where X is any residue and h is a hydrophobic residue, as a part of the Rossmann fold [17]. Hence, it was expected that like the C. kluyveri enzyme either of the T. indiensis LPDs would be able to reduce Fe(III) complexes which is consistent with the zymogram result ( Figure 1).

Thioredoxin Reductase.
Among the high-scoring matches obtained by MS of the protein mixture from band 2 the only T. indiensis protein likely to possess Fe(III) reductase activity is thioredoxin reductase (Trx). Thioredoxin reductase-like ferric reductase (FeS) as well as typical thioredoxin reductase (Trx) is also present in T. scotoductus SA-01 [4]. Both T. scotoductus enzymes can reduce Fe(III)-NTA complex although reduction by Trx is less efficient [4]. Magnetospirillum gryphiswaldense MSR-1 thioredoxin reductase FeR5 has also been shown to possess ferric reductase activity [3].  [4]. In fact, in case of T. scotoductus FeS absence of CXXC has been found to be advantageous with respect to Fe(III) reduction [4]. T.

T. indiensis
indiensis Trx also appears to be a thioredoxin reductase-like ferric reductase.

NAD(P)H-Nitrite
Reductase. MS with samples extracted from band 3 showed the presence of T. indiensis NAD(P)Hnitrite reductase which was the only protein in the mixture likely to be an Fe(III) reductase. InterProScan identified a FAD/NAD(P)-binding domain in the protein and suggested that it belonged to pyridine nucleotide-disulfide oxidoreductase family of proteins. BLAST analysis found significant sequence similarity of T. indiensis nitrite reductase to NAD(P)H-nitrite reductases from Clostridium tetani, Bacillus cereus, and Heliobacterium modesticaldum (data not shown). Domain analysis showed that all these nitrite reductases belonged to the pyridine nucleotide-disulfide oxidoreductase family. A search in the whole PDB archive found Pyrococcus furiosus nitrite reductase to be structurally similar to T. indiensis nitrite reductase (PDBeFold = 11.64, rmsd = 2.16). It is not known if any of these pyridine nucleotide-disulfide oxidoreductases could reduce iron. However, as described earlier, one pyridine nucleotidedisulfide oxidoreductase, C. kluyveri dihydrolipoamide dehydrogenase BfmBC, has been shown to reduce Fe(III) complexes of citrate, ATP, and ADP in an NAD(P)H-dependent manner [2]. The 3D structure of T. indiensis nitrite reductase was predicted by ESyPred#D using Novosphingobium 6 International Journal of Genomics aromaticivorans ferredoxin reductase ArR crystal structure (PDB ID 3LXD) as the template (Table 3). Sequence and structural similarity of T. indiensis nitrite reductase with C. kluyveri BfmBC were found to be moderate (Table 4). Hence, it is likely that T. indiensis NAD(P)H-nitrite reductase is responsible for the reduction of Fe(III)-nitrilotriacetic acid in the native gel as shown in Figure 1.

Thioredoxin Disulfide Reductase.
Finally, as identified with high Mascot score, the only T. indiensis protein in band 4 most likely to be a Fe(III) reductase is thioredoxin disulfide reductase (Tdr) ( Table 2). BLAST analysis found high sequence similarity of T. indiensis Tdr with T. scotoductus Trx and M. gryphiswaldense FeR5 ( Table 4). The 3D structure of T. indiensis Tdr was predicted by ESyPred3D using Mycobacterium tuberculosis thioredoxin reductase crystal structure (PDB ID 2A87) as the template (Table 3). 3D structural alignment of T. indiensis Tdr against T. scotoductus Trx and M. gryphiswaldense FeR5 carried out with both PDBeFold as well as DaliLite produced significant values (Table 4). Besides, like the other thioredoxin reductases, FAD and NAD(P)H binding domain of T. indiensis Tdr also contained redox-active site CATCD. Hence, the enzyme possesses the true characteristics of a thioredoxin reductase. In fact, T. indiensis Tdr has a greater sequence and structural similarity to T. scotoductus Trx and M. gryphiswaldense FeR5 than T. indiensis Trx. Therefore, it can be surmised that like the T. scotoductus and M. gryphiswaldense enzymes T. indiensis Tdr also reduced Fe(III) as evident from the zymogram (Figure 1).

T. indiensis
Tdr also showed 43% similarity (97% query coverage) with a thioredoxin reductase (TR) from the hyperthermophilic bacterium Thermotoga maritima [18]. Threedimensional structure of T. maritima was predicted using Salmonella enteric AhpF crystal structure (PDB ID 1HYU) as the template (Table 3). PDBeFold analysis also showed significant structural alignment between the two enzymes ( = 15.95 and rmsd = 1.90). Both enzymes have a typical FAD-binding motif GXGXXG near the N-terminus, a nicotinamide nucleotide-binding motif GGGXXA near the middle, and an active redox center CXXC [18]. Like thermophilic T. indiensis BSB-33, T. maritima is capable of growing as a respiratory organism when Fe(III) is provided as an electron acceptor and T. maritima cell suspension reduces Fe(III) [19]. However, whether T. maritima TR is involved in Fe(III) reduction has not yet been shown.

Similarity with Cr(VI) Reductases.
It has been reported earlier that like several other mesophilic and thermophilic microorganisms T. indiensis BSB-33 is capable of reducing Cr(VI) besides Fe(III) [8]. In fact, some bacterial proteins have demonstrated their capability to reduce both Fe(III) and Cr(VI) [20,21]. In Shewanella oneidensis outer membrane proteins MtrC and OmcA are involved in both Fe(III) and Cr(VI) reduction [20]. However, to the best of our knowledge, the only cytoplasmic enzyme that has been shown to reduce both Fe(III) and Cr(VI) is FerB from Paracoccus denitrificans [21]. Initially FerB was characterized as a ferric reductase [21]. Subsequently, it was found to be capable of reducing chromate as well and showed significant sequence similarity with known chromate reducers ChrR from Pseudomonas putida and YieF from Escherichia coli [21].
In Thermus scotoductus SA-01 a peripheral membraneassociated protein, identified as a dihydrolipoamide dehydrogenase (LPD) by sequence homology, has been shown to couple NAD(P)H oxidation to Cr(VI) reduction [16]. The enzyme contains a redox-active disulfide which is not present in ChrR [16]. Both T. indiensis LPDs were found to possess significant sequence similarity with the T. scotoductus protein (Table 5). T. scotoductus LPD 3D structure was also predicted using Geobacillus stearothermophilus dihydrolipoamide dehydrogenase crystal structure (PDB ID 1EBD) as the template (Table 3). Structural alignments of T. indiensis LPDs with T. scotoductus LPD gave highscore and rmsd (Table 5) and there was also considerable overlap between their 3D structures (Figures 3(a) and 3(b)). Multiple sequence alignment by ClustalW2 also identified in T. scotoductus LPD as well as T. indiensis LPDs FAD-and NAD(P)-binding Rossmann folds amino acid sequence motif GX 1-2 GXXG together with either GXXXG or GXXXA motifs where the first glycyl residue of these motifs and the third glycyl residue of the GX 1-2 GXXG motif are the same [22]. It is, therefore, quite likely that both T. indiensis LPDs would also reduce Cr(VI) in an NAD(P)H-dependent manner. Further, in D. desulfuricans a thioredoxin reductase MreD has been shown to be involved in NAD(P)H-dependent reduction of U(VI) and Cr(VI) in association with thioredoxin and possibly an oxidoreductase protein MreG [23]. The reduction mechanism as indicated consists of transfer of electrons from NAD(P)H to thioredoxin catalyzed by thioredoxin reductase [23]. Reduced thioredoxin could then transfer the electrons directly to oxidized metals or indirectly through MreG [23]. Three-dimensional structures of D. desulfuricans MreD, thioredoxin, and MreG and corresponding proteins of T. indiensis were predicted by ESyPred3D using PDB templates as enlisted in Table 3.
T. indiensis thioredoxin was found to contain the typical thioredoxin fold which is a distinct structural motif consisting of a four-stranded -sheet and three flanking -helices [24]. ClustalW2 alignment has shown that it also shared a CXXC active site motif. Structural alignment of T. indiensis and D. desulfuricans thioredoxins gave = 9.10, rmsd = 1.597 (PDBeFold) and = 13.5, rmsd = 1.7 (DaliLite). T. indiensis also contained a protein indolepyruvate ferredoxin oxidoreductase which was structurally similar to Desulfovibrio pyruvate ferredoxin/flavodoxin oxidoreductase MreG Sequence similarity between T. indiensis Tdr and D. desulfuricans MreE was found to be 37% with 97% query coverage. Structural identity between the two enzymes as shown by -score, rmsd, and superposition was very high (Table 5; Figure 3(c)). Besides, like the other thioredoxin reductases, T. indiensis Tdr also contained a redox-active site CATCD. Hence, based on the overall sequence and structural similarity, it could be inferred that like the thioredoxin-thioredoxin reductase system of D. desulfuricans, the thioredoxin-thioredoxin system of T. indiensis could also reduce Cr(VI). It is also to be noted that neither T. indiensis Tdr nor LPDs showed any significant sequence or structural match with ChrR, YieF, or FerB indicating the possibility of different chromate reduction mechanisms in this bacterium.

Conclusion
In conclusion, the soluble fraction of T. indiensis contains four distinct Fe(III) reductase activities. The annotated genome sequence of the thermophilic bacterium has enabled identification of the proteins responsible for Fe(III) reductase activity by chromatography and mass spectrometry. Two of these activities are due to pyridine nucleotide-disulfide family of enzymes, namely, two paralogous dihydrolipoamide dehydrogenases and NAD(P)H-nitrite reductase. A thioredoxin reductase-like protein is responsible for the third activity whereas the fourth activity is caused by a thioredoxin disulfide reductase having all the characteristics of a true thioredoxin reductase. Besides, dihydrolipoamide dehydrogenases and thioredoxin-thioredoxin reductase system of T. indiensis are also possibly responsible for chromate reductase activity of the bacterium. The genome sequence of T. indiensis will enable cloning and overproduction of the reductases identified in this work and further analysis to elucidate the similarities and differences of iron and chromium reduction pathways.

Conflict of Interests
The author declares that there is no conflict of interests regarding the publication of this paper.