Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines

Journal of Environmental Sciences 20(2008) 1348–1355

Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines CHANG Jin-Soo, REN Xianghao, KIM Kyoung-Woong ∗ Department of Environment Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju, Republic of Korea. E-mail: [email protected] Received 5 November 2007; revised 14 December 2008; accepted 12 March 2008

Abstract Biogeochemical cyclic activity of the ars (arsenic resistance system) operon is arsB influx/efflux encoded by the ecological of Pseudomonas putida. This suggests that studying arsenite-oxidizing bacteria may lead to a better understanding of molecular geomicrobiology, which can be applied to the bioremediation of arsenic-contaminated mines. This is the first report in which multiple arsB-binding mechanisms have been used on indigenous bacteria. In ArsB (strains OS-5; ABB83931; OS-19; ABB04282 and RW-28; ABB88574), there are ten putative enzyme, Histidine (His) 131, His 133, His 137, Arginine (Arg) 135, Arg 137, Arg 161, Trptohan (Trp) 142, Trp 164, Trp 166, and Trp 171, which are each located in different regions of the partial sequence. The adenosine triphosphate (ATP)-binding cassette transports, binding affinities and associating ratable constants show that As-binding is comparatively insensitive to the location of the residues within the moderately stable α-helical structure. The α-helical structures in ArsB-permease and anion permease arsB have been shown to import/export arsenic in P. putida. We proposed that arsB residues, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171 are required for arsenic binding and activation of arsA/arsB or arsAB. This arsB influx/efflux pum-ping is important, and the effect in arsenic species change and mobility in mine soil has got a significantly ecological role because it allows arsenic oxidizing/reducing bacteria to control biogeochemical cycle of abandoned mines. Key words: arsenic resistance bacteria; arsB influx/efflux pump; arsenic resistance system (ars) biogeochemical

Introduction Molecular biogeochemical cyclic activity of bacterial ars (arsenic resistance system) operons can be used to biomethylate arsenic (As) in arsenic-contaminated mines as the As-influx/efflux of this element usually results in mobility and toxicity (Chang et al., 2007, 2008; Bentley and Chasteen, 2002; Macalady and Banfield, 2002; Newman and Banfield, 2002). The environmentally molecular biogeochemical As is complex due to its participation in the arsenic reaction pathways of arsenic-resistant bacteria (Mukhopadhyay et al., 2002; Newman and Banfield, 2002). Arsenic resistance systems affect how arsenic resistant bacteria mediates gene expression and biogeochemical transformations, thereby play a key role in the biogeochemical cycles of arsenic-contaminated mine (Oremland et al., 2004, 2005; Saltikov et al., 2003; Santini et al., 2000; Silver and Phung, 2005). Arsenic resistant gene expression systems allow arsenic methylation through various bacteria and contain both a chromosome gene and a plasmidencoded arsenical operon (Bruhn et al., 1996; Chen et al., 1996; Ji et al., 1994; Silver and Keach, 1982). This suggests that a change in the arsenic redox state or species by ars containing bacteria has got significantly important ecological role and may lead to a better understanding * Corresponding author. E-mail: [email protected].

of molecular geomicrobiology. Arsenic resistant systems (ars) have a variety of operons. The diversity in the representative arsenic resistance operon systems shown includes gene operons, arsenite oxidizing and arsenate reduction genes. Arsenic resistance systems and the corresponding mechanisms have been reported (Fig.1) (Chen and Rosen, 1997; Kostal et al., 2004; Kurdi-haidar et al., 1996; Kuroda et al., 1997; Lin et al., 2006; Lee et al., 2001; Malasarn et al., 2004; Meng et al., 2004; Qin et al., 2006; Rosen, 1999; Tisa and Rosen, 1990; Wang et al., 2000; Wu et al., 1992; Zhou et al., 2000). Arsenic resistance systems as ars ATPase have been attracting attention with respect to the inner membrane as well as cytoplasm and classes of transporters, e.g., ArsA/ArsB efflux/influx pumps (Kuroda et al., 1997; Lin et al., 2006). Pumping mechanisms of the ArsA and ArsB proteins forms an arsenite/arsenate efflux/influx ATPase. Bhattacharjee and Rosen (1996) and Ruan et al. (2006) reported that Cystenine (Cys)-113, Cystenine (Cys)-172 and Cystenine (Cys)-422 form a high affinity metalloid binding site in the ArsA ATPase, which is arsenite/antimony-stimulated ATPase; the catalytic subunit of the ArsAB coupling efflux/influx pump. The arsB gene is on the inner membrane and functions alone as an arsenic transport, whereas the arsA codes an unique ATP pump and binds arsenic/antinomy. A report evaluated with 429 residues or 12-helix protein Trp 16, Trp 24, His 20

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Fig. 1 Overview of membrane associated uptake, efflux/influx, oxidation and reduction of arsenic ion. Arsenic is given more emphasis, as enzymes in the periplasm and cytoplasm are included as well as classes of transporters ArsA/ArsB ATPase efflux/influx pump. The arsB efflux transport systems extend from the cytoplasm across the outer membrane of Gram-negative bacteria. How the substrates affect the efflux/influx systems, that do not have associated with outer membrane protein function, is not known. arsM: arsenite S-adenosylmethyltransferase evolved to methylate arsenic; arsR: firstgene of the lead operon; arsD: second-gene of the lead operon; arsA: arsenic-stimulated ATPase; arsB: arsenic influx/efflux pump and inner membrane; arsAB: arsenic strong pump of coupling influx/efflux and an anion-translocating ATPase; arsC; arsenate to arsenite, reductase gene; arsH: unknown operon of arsenite-oxidizing/reducing; arrA and arrB: arsenate reductase gene; aoxA, aoxB, aoxC, aoxD, aroA and aroB: arsenite oxidation gene.

and Arg 23 of the arsenic binding domains required for delivery of As(III) to and activation of the ArsB pump (Broome-Smith et al., 1999; Dey and Rosen, 1995). The arsB-mediated transformation is proposed to have an important influence on arsenic cycle of arsenic-contaminated mines. This study shows that the isolated indigoenous bacteria whose habitats were arsenic-contaminated soils, mostly had the molecular biogeochemical arsB gene for arsenic resistance. The data suggests that three enzyme types of the arsenite/arsenate-binding site model formed by the efflux/influx are required for biogeochemical cyclic activity.

1 Materials and methods 1.1 Cultivation of arsenite-oxidizing bacteria decision One gram of tailing and 1 g of sediment were collected for isolating pure bacteria from 3 sampling sites at two abandoned silver and gold mines areas (Myoung-bong and Duck-um) in South Korea. 5 g of each tailing and sediment was extracted for As analysis by shaking for 1 h, and digested in aqua regia (4 ml) consisting of HNO3 and HCl (1 ml : 3 ml) and heated at 70°C on a shaker for 1 h. 16 ml of deionized water was then added to the solution (Ure, 1995). Arsenic content were determined by HGAAS (hydride generator-atomic absorption spectrometry, Perkim-Elmer ZL5100, Waltham, USA) and Flame-AAS (Perkim-Elmer ZL5100, Waltham, USA). The tailing and sediment samples were added to MSB (Stranier’ Basal Medium) medium containing sodium arsenite (NaAsO2 , Sigma, USA) and sodium arsenate (Na2 HAsO4 , Sigma, USA) (Chang et al., 2007; Stanier et al., 1966). After several transfers, the isolated colonies were assessed for

arsenic(III) and arsenic(V). Arsenite-oxidizing bacteria were cultured at 30°C in MSB (pH 7) with 1 mmol/L D(+)glucose and ethanol as carbon sources. Strains grew in MSB, which was prepared as follows: 4.8×10−4 mmol/L Na2 HPO4 ·7H2 O (Sigma, USA), and 1.5×10−4 mmol/L KH2 PO4 (Sigma, USA), 1.7×10−4 mmol/L NaCl (SigmaAldrich, USA), 1.8×10−2 mmol/L KNO3 (Junsei, USA), and 5 ml of 1.5×10−4 mmol/L (NH4 )2 ·SO4 (Sigma, USA). The solution were mixed and the volume was adjusted to 1 L with distilled water. The As(III) and As(V) were used at concentrations from 0 to 66.7 mmol/L and 133.4 mmol/L, respectively by the 7th pure culture. Indigenous bacteria were used for fluorogenic and/or chromomeric reaction (Manafi et al., 1991). The panel was then placed within the BBL CRYSTALTM identification system (Bescton, Dickoinson and Company, MD, USA) and incubated at 37°C for 20 h. To test the ability of the strains to oxidize arsenite, the isolates were inoculated in 250-ml glass flasks with 60 ml of MSB and 75 mg sodium arsenite. To monitor oxidation of As(III) to As(V) over the period of incubation, the As(III) concentration was determined by measuring the amount of As(V) using a silica-based strong anion cartridge (LC-SAX SPE, Supelco, Belle-Fonte, USA) (Le et al., 2002). The concentration of As(III) and As(V) was measured using a hydride generation atomic absorption spectrophotometer (HG-AAS, Perkin Elmer ZL 5100, Waltham, USA). 1.2 SEM analysis To prepare scanning electron microscopy (SEM) specimen for arsenite-oxidizing bacteria cell association, cells were fixed in 1.5% gutaradehyde in 0.0067 mol/L phosphate-buffered saline (PBS; pH 6.5) at 4°C overnight

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and washed with the DI water (Oremland et al., 2002). The sample analysis was performed with an S-4700 FE-SEM (HITACHI, Tokyo, Japan) operating at 5 kV. 1.3 PCR amplification of arsB gene Genomic DNA of arsenite-oxidizing bacteria was prepared from the bacterial cultures by following standard methods (Sambrook and Russel, 2001), and using a 1-ml microcentrifuge tube with the appropriate individual colony. Genomic DNA was extracted from every arsenite-oxidizing bacteria colony (107 colony forming units (CFU)/ml) with 1 ml of TES (10 ml Tris-HCl, 50 mmol/L EDTA, 10% sodium dodecyl sulfate) and 10 μl of proteinase K (50 mg/L), then allowed to react in a 55°C tremulous cistern for 10–12 h to digest the protein. Genomic DNA had an absorbance ratio (A260 nm /A280 nm ) ranging from 1.7 to 2.0. PCR was conducted in 50 μl total volume (0.5 g genomic DNA; 10 pmol primer) containing the following: Taq DNA polymerase(5 U/g), 10× PCR buffer (Mg2+ ), 100 mmol/L Tris-HCl pH 8.3, 500 mmol/L KCl, and 15 mmol/L MgCl2 ), and dNTP mixture. The coding for arsenite-oxidizing genes was determined by PCR using universal sense-primer SEL0904 (5’-ATC ATG GCT CAG ATT GAA CGC-3’) and antisense-primer SEL1226 (5’-T ACC TTG TTA CGA CTT CTA CCT-3’). ArsB pump uses primers for AGL0929 ArsB sense (5’-GTG GAA TAT CGT CTG GAA TGC GAC-3’) and AGL0226 ArsB Anti-sense (5’-GGT AAT TTT CGG CCC CAA ATC G-3’). PCR amplification (35 cycles of denaturation at 94°C for 5 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, followed by an additional extension for 7 min at 72°C) was performed in 10 pmol of each primer. PCR was conducted in a Mastercycler Gradient (Eppendorf, Germany), and the PCR products were identified by gel electrophoresis using 0.7%–1.5% agarose gels. The samples were sequenced by an automated DNA sequencer (Model 3100, ABI PRISM Genetic Analyzer System Profile, Korea). 1.4 Phylogenetics analysis and ArsB structure modeling of arsB-related pump alignment Sequencing was performed in a 6-μl volume with 15–50 ng of PCR product and 1 pmol of each primer. The samples were analyzed using an automated DNA sequencer (Model 3100, ABI PRISM Genetic Analyzer System Profile, USA). The sequencing results of 16S rDNA arsB were compared for homology of the NCBI (National Center for Biotechnology Information, USA) database. The 16S rRNA and arsB sequences were compared with known sequences in the NCBI gene database. BLAST alignment was integrated for amino acid homology of Vector NTI Suite v5.5.1 (InforMax, USA). Database sequences with fewer than 1,500 nucleotides were excluded from the phylogenetic and arsB analysis homologies. GeneBank accession numbers were received for 16S rRNA/arsB pumping partial from NCBI to Pseudomonas putida strain OS-5 (16S rRNA; DQ273565 and arsB; ABB83931), P. putida strain OS-19 (16S rRNA; DQ223972 and arsB; ABB04282), and P. putida strain RW-28 (16S rRNA;

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DQ279763 and arsB; ABB88574). The arsB-binding sequence was manually aligned to increase the similarity. The iterative method of MODELLER 8v1 (Lutfullah et al., 2007) was carried out to get a good model (Marti-Renom et al., 2000). Arsenic enzymes of arsB-binding predicted model were arsenite/arsenate bacteria, which might provide an understanding of molecular geomicrobiology.

2 Results and discussion 2.1 Selection and characterization of arsenite-oxidizing bacteria Sampling point of arsenite-oxidizing bacteria in arsenic concentration was found in both the tailings and sediments from the Myoung-bong (strain OS-5; 26.90 mg/L) and Duck-um (strain OS-19; 54.7 mg/L and strain RW-28; 83.9 mg/L) mines (Chang et al., 2007). It was first reported by Chang et al. (2007) that arsenite-oxidizing bacteria was recognized as an arsB-binding enzyme capable of supporting growth of indigenous species. Some researchers have reported arsenite-oxidizing/arsenate-reduction in arseniccontaminated mines or environments by diverse bacteria, which use arsenic or glucose carbon sources as the oxyanion-translocating pump for their geochemical cyclic activity (Tisa and Rosen, 1990; Wu et al., 1992; Zhou et al., 2000; Suzuki et al., 1998; Neyt et al., 1997; Bruhn et al., 1996; Rosen et al., 1999) (Table 1 and Fig.1). The ArsA-B-AB enzyme is involved in arsenic bacteria oxidation and reduction. However, according to Table 1, similar isolates have also been found in arsenic contaminated mine tailing/sediment. Most of the previously described arsenic pumping bacteria were heterotrophic, and the most common isolated was P. putida of indigenous (Chang et al., 2007), plasmids type (Table 1) genotype of arsA-B-AB by pumping homologues contributes to the mine cycle of arsenic-contaminated mine. Thus, arsenic pumping may be a protective mechanism for individual arsA-B-AB and an important link in the arsenic mobilization. The Ars pumping protein is a membrane enzyme that functions alone as a chemi-osmotic arsenic transport (Table 1). Table 1 and Fig.1 summarized the arsB efflux/influx pump arsenicrelated gene in which we proposal the arsenic mobile enzyme. Fig.1 is an overview of membrane associated uptake, efflux/influx, oxidation and reduction of arsenic. The arsA-B-AB efflux/influx pumps arsenic mobile system from cytoplasm across the outer membrane of prokaryotes bacteria. It is still unknown how the substrates in the pumping system that do not have indicated associated outer membrane protein function. Bacteria ars operons encode an arsenic efflux/influx system that can be a protein carrier (ArsA-ArsB) or an anion translocating ATPase (ArsAB). Fig.1 shows the arsenic mobile and specie change of As-contaminated mines type. Prokaryotes have efflux/influx types of arsenic (sodium arsenite, sodium arsenate, monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA)) transporters, arsA-B or arsAB. The best-characterized lead gene activity was encoded by arsRarsD operon (Fig.1) and arsenite oxidation gene of aoxA,

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Table 1 Strains and plasmids used in this study Strain/plasmid

Accession no.

arsAB genotype, description

Source

Pseudomonas aeruginosa E. coli RW-29 E. coli R773 E. coli pUM3

ABB016022

J02591

PDBb Chang et al., 2007 Tisa et al., 1990 Chen et al., 1996

E. coli R773 E. coli

IF48b

arsB arsB, anion pump, Duck-um Mine sediment arsA and arsB ATPase activity, anion pump arsA and arsB binding site (AGGAGG), arsB 423 bp, polar, non polar arsB, 12 inner membrane-spanning α-helices arsAB (His 148, Ser 420, Cys 113, Cys 172, Cys 422, His 453) pump ATPase arsB 429 bp, plasmid pKW301, acid mineral water arsB efflux pump, 1,289 bp, pI258 arsB, efflux pump, 1,283 bp arsA (Cys 113, Cys 172, Cys 422) arsB, Duck-um Mine sediment arsB, plasmid JM109 arsAB (Cys 113, Cys 172, Cys 422), ATP-coupled anion pump arsB, similar to E. coli plasmid R773 ArsB arsB, arsenite membrane pump arsB, Myoung-bong Mine sediment arsB, Duck-um Mine tailing arsB, Myoung-bong Mine tailing arsB, Duck-um Mine tailing arsB (His 133, Arg 137, Trp 166), anion pump, Duck-um Mine sediment arsA, arsenite-stimulated ATPase, arsB, membrane transporter arsB (His 138, Trp 142, Trp 171), Duck-um Mine tailing arsB, Myoung-bong Mine sediment arsB, Myoung-bong Mine sediment arsB (Trp 16, His 20, Arg 23, Trp 24), anion pump, Duck-um Mine sediment arsB, Duck-um Mine sediment arsB (His 138, Trp 142, Trp 171), anion pump, Myoung-bong Mine tailing arsAB, pump-driving ATPase, bacteriphage M13 arsB, Duck-um Mine sediment arsB, Myoung-bong Mine tailing arsB, Duck-um Mine sediment arsB, arsenite pump arsB, oxyanion-translocating pump, arsA, arsenite activated ATPase arsAB, arsD catalytic subunit

Acidiphilium multivorum AIU301 Staphylococcus aureus Pseudomonas putida TP3 E. coli JM109 Pseudomonas putida OW-18 E. coli DH5-α E. coli

AB004659 M86824 AJ271973 DQ141542

Shewanell sp. ANA-3 Alcaligenes faecalis Pseudomonas rhizospherea OW-2 Pseudomonas putida OW-16 Pseudomonas putida RS-4 Pseudomonas putida RS-17 Pseudomonas putida RW-28

AY271310 AY297781 AY866408 DQ112328 DQ112332 DQ112329 DQ112333

Klebsiella oxytoca pMH12

AF168737

Pseudomonas putida OS-19 Pseudomonas putida OW-4 Serratia marcescens RW-5 E. coli OS-80

AY866406

ABB76686

Vibrio ichthyoenteri RW-29 Pseudomonas putida OS-5

AY952321

E. coli R773 Pseudomonas putida OW-20 Pseudomonas putida RS-5 Pseudomonas putida RW-26 Yersinia enterocolitiva Tn2502 IncN pR46 E. coli JM109 a

J02591

DQ141544 U58366 AY046276

Wu et al., 1992 Zhou et al., 2000 Suzuki et al., 1998 Ji et al., 1994 NCBIa Bhattacharjee and Rosen, 1996 Chang et al., 2007 Chang et al., 2007 Rosen et al., 1999 Saltikov et al., 2003 NCBIa Chang et al., 2007 Chang et al., 2007 Chang et al., 2007 Chang et al., 2007 This study NCBIa This study Chang et al., 2007 Chang et al., 2007 Chang et al., 2007 Chang et al., 2007 This study Chen et al., 1986 Chang et al., 2007 Chang et al., 2007 Chang et al., 2007 Neyt et al., 1997 Bruhn et al., 1996 Lin et al., 2006

http://www.ncbi.nlm.nih.gov/ (National Center for Biotechnology Information). b http://www.rcsb.org/pdb/home/home.do (Protein Data Bank).

aoxB, aoxC, aoxD, aroA, and aroB in bacteria. Prokaryotes extrude arsenic using YqcL, ArsB, ArsAB, and Ars. A membrane carries protein or sequesters it in vacuoles as the 3-glutathione transporter. On the basis of the analysis of arsenite-oxidizing bacteria, different isolates were selected and substrates characterization of these isolates was done. Biochemical and physiological features of three isolates are reported in Table 2. Arsenite-oxidizing bacteria isolates were rod shaped, thin gray, 0.8×2.0 μm size, and at 30°C. The indigenous bacteria strains OS-5 (pH 6.1), OS-19 (pH 4.0), and strain RW-28 (pH 7.0), and Eh (mV) of sampling site strains OS-5; 300 mV, OS-19; 194, and strain RW-28; 172 were assessed at 2 arsenic-contaminated pond sites and 2 arsenic tailings located in the Duck-um mine and Myoung-bong mines. Physiological and biochemical tests of the isolated strains demonstrated that arsenite-oxidizing of the isolated bacteria revealed very similar properties to each other and had an opposite activity for the positive substrates: sucrose, adonitol, p-n-p bis-phosphate, p-n-pβ-glucuronide, urea, glycine, citrate, triphenyl, ethanol,

and glucose (Table 2). The arsenite-oxidizing bacteria belonging to the γ-proteobacteria has the three different phylogenetic affiliations. According to Chang et al. (2007), arsenite-oxidizing bacteria isolated from the Ascontaminated mines included species with a low G+C content: strain OS-5 (44%), strain OS-19 (50%), and RW-28 (52%). Arsenic species can change the geomicrobiological population of habitable mine areas, throught their metabolic physical activites, therefore, affect the cyclic biogeochemical properties of arsenic-contaminated areas. It also seems that the arsB metalloenzymes from microorganisms found in abandoned mines often have unusual arsenic efflux/influx pumping functions. Arsenic or transition element that can be used by bacteria arsB genotype function may actually be used in geochemical As-pathways by microorganisms abandoned mines. 2.2 arsB genotypes and characterization of ArsB binding-structural The ars genotype has already proven useful PCR product screening of the different size strains, OS-5; 914

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Table 2 Biogeochemical and physiological properties of Pseudomonas putida by the arsenite-oxidizing strain OS-5, RW-28, and OS-19 Characterization/substrates Strain OS-5b

Strain RW-28 Strain OS-19

Accession number Arsenic contamination of sampling sitea pH of sampling site Eh of sampling site (mV) Gram staining Doubling time (h) Shape Color of colony Motility Size Temperature range for growth Optimum temperature for growth Glucose Ethanol Arabinose Mannose Sucrose Melibose Rhammose Sorbitol Mannitol Adonitol Galactose Inositol p-n-p-Phosphate p-n-p α-β-Galactoside Proline nitroanilide p-n-p bis-Phosphate p-n-p-Xyloside p-n-p-α-Arabinoside p-n-p-β-Glucuronide p-n-p-N-actyl Glucosaminide γ-L-Glutamyl p-nitroanilide Esculin p-nitro-DLPhenylalanine Urea Glycine Citrate Malonic acid Triphenyl terazolium chloride Arginine Lysine

ABB83931 26.00 ± 17.9

ABB88574 54.79 ± 15.1

ABB04282 83.90 ± 2.3

6.1 300 – 18 rod Thin gray + 1.1×2.0 μm 25–37°C

5.5 172 – 20 rod Thin gray + 0.8×2.0 μm 25–37°C

4.2 194 – 25 rod Thin gray + 0.8×2.0 μm 25–37°C

25–32°C

25–32°C

30°C

+ + – + – + – – – + + – – – + + – – + +

+ + + – – + – + – + + + + + – + – – + +

+ + – + – + + + – + + + + + + + – – + +

+

–

+

– –

+ –

– –

+ + + + +

+ + + + +

+ + + + +

– –

– –

+ +

a Results in mg/kg (means±standard deviation). b Control strains: P. putida strain OS-5 (AY952321). +: positive; –: negative. n=3.

bp, OS-19; 904 bp, and RW-28; 897 bp, from arseniteoxidizing bacteria (Fig.2). Therefore, arsB type diversity should be considered when using biogeochemical genotype PCR-based and plasmids-based vectors to examine position in the inner membrane (Bentley and Chasteen, 2002; Bruhn et al., 1996; Macalady and Banfield, 2002; Ji et al., 1994; Newman and Banfield, 2002; Mukhopadhyay et al., 2002; Suzuki et al., 1998). The studies found that the energy-dependent efflux mechanism of bacteria oxidation/reduction to molecular characterization properties of the ArsB protein anion pump is in the membrane (Silver and Keach, 1982; Silver and Phung, 2005; Tisa et al., 1990). Biogeochemical cyclic activity of ars oper-

Fig. 2 Agarose gels 0.7% showing the PCR products are amplified from the genomes of several arsenite-resistance bacterial strains. Land M: Lambda DNA/HindIII size mark (Promega, USA). The strains shown are OS-5 (control), OS-19 and RW-28.

on is arsB influx/efflux encoded by the ecological ars of arsenic-contaminated abandoned mines areas (Fig.1). This information proposed that arsenite-oxidizing bacteria might lead to the understanding of arsenic species and studies of molecular geomicrobiology. This is the first report in which multiple arsB mechanisms have been used to study indigenous bacteria. The arsB efflux/influx is correct. The pumping plays a very important ecological role in arsenic toxicity and mobility in abandoned mine areas, as it facilitates the biogeochemistry cyclic activity of arsenite-oxidizing bacteria. The arsB genotype of arsenic-binding P. putida strains, OS-5; ABB83931, OS-19; ABB04282, and RW-28; ABB88574 has ten putative amino acid, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171. Everyone located in different regions of the partial sequence. According to Bhattacharjee and Rosen (1996) and Ruan et al. (2006) spatial proximity between Cys 113, Cys 172, and Cys 422 in the metallocativate domain of the arsA ATPase, Cys 113, and Cys 422 form a high affinity metalloid binding site in the arsA ATPase (Table 1). The coupling ArsAB ATPase-binding cassette transport, binding affinities and association rate constants show that arsenic species change is comparatively insensitive to the location of residues within moderately stable α-helical structure (Fig.4). The α-helical structures ArsB-permease and anion permease arsB have been shown to import/export arsenic in indigenous bacteria of arsenic-contaminated mine areas. Table 1 shows the arsB-binding genotype of the bacteria/plasmids and other enzyme sites of them carrying the ATPase pump residues: Cys 113, Cys 172, Cys 422, and His 453; coupled anion pump residues: Cys 113, Cys 172, and Cys 422 and polar type sequence: AGGAGG (Bhattacharjee and Rosen, 1996; Chen et al., 1996; Rosen, 1999). In this study, the presence of an arsB influx/efflux pump system encoded by the arsB homolog sequences was found in all 3 isolates (Figs.3 and 4). Lin et al. (2007) proposed that arsD

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Fig. 3 Sequence alignments of the arsenite-oxidizing bacterial to proteins of the arsB family. The putative arsenic-binding is boldface. P. aeruginosa (ABB016022). P. putida strain OS-5 (ABB83931); P. putida strain OS-19 (ABB0428); Pseudomonas putida strain RW-28 (ABB88574).

Fig. 4 Modeling structure of the arsB efflux pump protein. Ribbon diagram of the active site of arsenic, shown in yellow, shown in white of arsenic binding enzyme, respectively. (a) OS-5; (b) OS-19; (c) RW-28; (d) arsB influx/efflux. In Fig.4d, schematic representation of arsenic binding to a marginally stable helical peptide containing four Trp, three His and three Arg. SEM images of arsenite-oxidizing bacterial OS-5 (AY952321), RW-28 (DQ112333) and OS-19 (AY866406) are shown. SEM image illustrate that the cell of strain P. putida are curved rods measured 0.2–1.0 μm in length and 1.0–5.0 μm in width.

residues, Cys 12, Cys 13 and Cys 18 but Cys 112, Cys 113, Cys 119, or Cys 120, are required for delivery of As(III) and activation of the ArsAB (Lin et al., 2007). In addition, ArsA is an arsenite/antimony-stimulated ATPase, the catalytic subunit of the coupling ArsAB extrusion pumping (Bhattacharjee and Rosen, 1996). The As-binding possibilities predicted from structures in the Protein Data Bank (www.rcsb.org/pdb/home/home.do) suggested that the arsenic binding activity of novel arsB bacteria site could be used for protein modeling of molecular geochemistry of metalloid binding. We proposed that arsB residues, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171 are required for arsenic binding to activate of the ArsA/ArsB or ArsAB. This ArsB influx/efflux pumping is correct and the change in arsenic species, arsenic toxicity and mobility in mine soil has a very important ecological role because it allows arsenic oxidizing/reducing bacteria to control biogeochemical cycle of abandoned mines.

3 Conclusions This study described the first investigation into the efflux/influx pump of arsB-binding genes presented in indigenous bacteria. Arsenic contamination of tailing/water/sediment supplies is a problem, which can cause ecological damage in abandoned mines. The proposal for the substrate of ArsB protein explains the present results of that As-bindizing site involves 10 enzymes. We believe that genotype modeling of arsenic protein and arsB-binding enzyme research of the arsenic-contaminated mine areas significantly improved our understanding of the mine restoration management. Protein modeling of ArsB suggests that indigenous bacteria can influence arsenic speciation in natural settings of arsB geochemistry cyclic activity because their presence predicts arsenic redox. However, further studies must be conducted to understand the role of ars genotype of the arsB-binding enzyme in the molecular geochemistry cyclic activity found in

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arsenic-contaminated abandoned mine areas. The ArsB model implies that the As-binding site is in the change of arsenic species, although the bacteria arsB is in arseniccontaminated mines. Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Korean Ministry of Science and Technology (No. M10300000298-06J000029810).

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