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Bacterial aoxB gene encoding arsenite oxidase was identified from As(III)-oxidizing bacteria isolated from from gold mine tailings of Xinjiang, China which is worst affected with arsenism. It was speculated that As(III)-oxidizing bacteria isolated from those highly arsenic-contaminated areas contributed the biogeochemical cycling of arsenic by transforming arsenic species and resulting in change of mobility. XS3 strains exhibited high As(III) (1000 mg/l) resistance. This strains oxidized 6400 mg/l As(III) in sucrose minimal salt low phosphate medium (SLP) medium. The ability of strains to resist high concentration of arsenic and oxidize As(III) efficiently which makes them potential candidates for bioremediation of arsenic contaminated environment. Arsenite oxidase activity was found in both fraction supernatant as well as in spheroplast preparation. Based on 16s rRNA gene sequence analysis, strain XS3 was closely related to genus Pseudomonas thivervalensis.
Keyword: aoxB gene; Arsenite oxidase; Spheroplast preparation; Pseudomonas thivervalensis; Bioremediation
Arsenic is present in various environments, is released either by natural weathering of rocks or by anthropogenic sources (e.g., mining industries and agricultural practices) and is found in the oxidation states +5 (arsenate), +3 (arsenite), 0 (elemental arsenic), and -3 (arsine). Contamination of drinking water supplies with the inorganic soluble forms arsenite and arsenate has often been reported, and arsenic has been identified as a major risk for human health in different parts of the world (northeast India, Bangladesh, China, northwest United States) (Pontius et al., 1994; Sun, 2004). USEPA (2001) has lowered the MCL (maximum contaminant level) of arsenic from 50 to 10 Î¼g/l in drinking water. High levels (â‰¥ 500 mg/l) of arsenic concentration have been detected in several metalliferous manufacturing industries such as copper and gold smelter (Basha et al., 2008). The biogeochemical cycle of this element strongly depends on microbial transformation, which affects the mobility and the distribution of arsenic species in the environment (Quinn and McMullan, 1995). Faulwetter et al. (2009) described removal of a particular pollutant is typically associated with a specific microbial group. Chaturvedi et al. (2006) found fifteen bacterial isolates from effluents successively removed heavy metals from distillery effluents. Some studies like Ertugrul et al. (2009) found heavy metal bioaccumulation using yeast cells.
Several bacteria involved in transformation processes comprising reduction, oxidation, and methylation of arsenic species have been described (Cullen and Reimer, 1989; Dowdle et al., 1996; Newman et al., 1998; Salmassi et al., 2002). The toxicological effects of arsenic are related to its chemical form and oxidation state; the organic forms are the less toxic. Among inorganic forms, As(III) is reported to be on average 100 times more toxic than the less mobile As(V) (Neff, 1997). Several remediation processes have been described for arsenic removal based on chemical oxidation of arsenite to arsenate followed by alkaline precipitation (Bothe et al., 1997; Gregor, 2001; Hering et al., 1997). The major drawbacks of these processes are that they generate additional pollution and are expensive. This has led to the exploration of alternative methods for arsenic remediation based on its biological oxidation. Several arsenite-oxidizing bacteria have been isolated, starting with an Achromobacter strain in 1918 (Green, 1918). Since then, different arsenite-oxidizing bacteria, including Pseudomonas strains (Ilyaletdonov and Abdrashitova, 1981), Alcaligenes faecalis (Osborne and Enrlich 1976; Philips and Tylor, 1976) Thiobacillus ferrooxidans and Thiobacillus acidophilus (Leblanc, 1995) bacteria from the Agrobacterium/Rhizobium branch of Î²-Proteobacteria (Santini et al., 2000) and bacteria of the Thermus genus (Gihring et al., 2001) have been described.
Some bacteria show resistance to arsenite through the activity of an arsenite oxidase (Muller et al., 2003) or resistance to both arsenite and arsenate through the genes of the ars operon. The latter is a genetic system that consists of a regulatory gene (arsR), an arsenite specific transport protein that removes arsenite from the cell (arsB), and an arsenate reductase (arsC) that reduces arsenate to arsenite. Cai et al. (2009) described arsenite oxidase gene clusture (aoxX-aoxS-aoxR and aoxA-aoxB-aoxC-aoxD) which was involved in arsenic redox transformation by arsenite oxidizing bacteria in Achromobacter and Pseudomonas sp. Present research focused on the finding of indigenous efficient bacterial species in arsenic-contaminated sites and its characterization for improvement of arsenic bioremediation.
2. Materials and Methods
2.1. Isolation, enrichment and screening
Sample was collected from the gold mine tailing area of Karamay, Xinjiang, China. Mine tailing sample (5 g) was inoculated in the 250-ml conical flask containing 50 ml sucrose minimal salt low phosphate medium (SLP) Sucrose 1%; (NH4)2SO4 0.1%; K2HPO4; MgSO4 0.05%; NaCl 0.01%; Yeast extract 0.05% and 5 mM of As(III). Flask was incubated on orbital shaker at 30Â°C for 5 days, and 3 ml of this enrichment culture was inoculated in fresh medium. This procedure was repeated twice and arsenic resistant bacteria were isolated by plating enrichment cultures on SLP containing 200 mg/l of As(III). Morphologically different colonies were isolated and purified by repeated sub-culturing on SLP with 200 mg/l As(III). One efficient culture were selected and designated as XS3. The cultures were routinely maintained on SLP with 100 mg/l of As(III). The glycerol stocks (20%) were also prepared and maintained at -20Â°C. As(III)-resistant strain obtained were screened for their As(III)-oxidation ability by using silver nitrate (AgNO3) screening method described by Simeonova et al. (2004) with slight modification. The strain were grown on SLP with 100 mg/l As(III) for 2 days at 30Â°C. The plates were flooded with AgNO3 solution (0.1 M). Brown precipitate around the colony indicated positive As(III) oxidation reaction. The colonies showing positive As(III) oxidation were further studied.
2.2. Identification of bacterial isolate
16S rRNA gene sequence and phylogenetic analysis were done by extracting genomic DNA from overnight grown cultures by a simple lysis protocol, as described in Kapley 2001. One microliter of purified genomic DNA served as template. 16S rRNA gene was amplified using MG25+ LongGene PCR systems (Scientific institute co. ltd, China) with the following set of primers: forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer: 5'-ACGGGCGGTGTGTTC-3' as described by Weisburg et al., 1991. Reaction mixture for the PCR contained 1X PCR buffer; 200 ÂµM of dNTPs; 1.5 mM MgCl2, 0.1 ÂµM of each primer and 2.5 units of Taq DNA polymerase (Invitrogen, USA) in a final volume of 100 Âµl sterile MQ water. The PCR was performed with an initial denaturation at 92Â°C for 2 min followed by 36 cycles at 92Â°C for 1 min, 48Â°C for 30 s and 72Â°C for 2 min and a final extension of 72Â°C for 6 min. The amplification product was gel purified using QIA-Gel extraction kit (Qiagen, USA), and ligated into the pGEM-T easy vector as per manufacturer's instructions (Promega Inc., USA). Ligated plasmid was transformed into E. coli DH5Î± cells by calcium chloride treatment and heat shock method. After screening of the positive clones, the partial sequence was generated by chain termination method using an Applied Biosystems (ABI Prism 3730 DNA analyzer) sequencer (DNA sequencing facility, Beijing Genomics Institute Beijing, China). The sequence was compared against the available DNA sequences from type strains in Gen-Bank (http://www.ncbi.nlm.nih.gov/) using BLASTN sequence match tool. The sequences were aligned using MultAlin (http://bioinfo.genotoul.fr/multalin/ multalin.html) program and the alignment was manually corrected and phylogenetic tree was constructed using the MEGA 4.0 (Tamura et al., 2007) software.
2.3. Determination of As(III ) resistance
Resistance of strain XS3 for As(III) was determined by growing them in SLP liquid medium amended with increasing concentrations of As (III) (from 100, 200, 400, 800, 1600, 3200, 6400, 1000 mg/l). Two flasks for each concentration were inoculated with appropriate cell suspension grown in SLP without arsenic to obtain initial optical density of approximately 0.02. The growth was evaluated by measuring the OD600 after 48 hour incubation at 30Â°C.
2.4. Arsenic transformation by XS3
To test for the ability to oxidize arsenite, XS3 were inoculated into 50 ml of growth medium containing 100, 200, 400, 800, 1600, 3200, 6400 and 1000 mg/l of arsenite and incubated at 30Â°C with 120 rpm shaking. Control experiments using noninoculated, sterile media with 100, 200, 400, 800, 1600, 3200, 6400, and 1000 mg/l arsenite were also incubated under the same conditions. The high initial level of arsenite used in these experiments was to better facilitate rate calculations and to confirm to analytical constraints. Two milliliter samples from biological and abiotic experiments were taken over time for measurements of cell density and for determinations of arsenic speciation. Optical density was measured at 600 nm using a DU*800 (Beckman Coulter-USA) UV/vis spectrophotometer. Samples were centrifuged and decanted, acidified by adding concentrated trace metal-grade HCl to 1% (v/v), and stored at 4Â°C for less than 7 days prior to arsenic analyses. Measurements of arsenic speciation in laboratory experiments followed by hydride generation atomic flame spectrometer (AFS-800, Beijing Titan Instrument. Co. Ltd. Beijing, China). For total As determination, it is necessary to acidify with 10 ml of conc. HCl and added 10ml of 10% (w/v) thiourea-ascorbic acid solution, diluted to 50 ml with ultrapure water, and then heated at 95oC for 30 min. Digested sample solutions were analyzed by AFS.
2.5. Induction of arsenite oxidase system
To check if the arsenite oxidase system was constitutive or inducible, strain were first grown in SLP without arsenic and sub-cultured three times in same medium. After third subculture, cells were inoculated separately into SLP without arsenic and with 100 mg/l As(III). After overnight incubation at 30Â°C, cells were centrifuged at 10,000 rpm at 4Â°C for 15 min and washed twice with saline solution. The cells were suspended in 20 ml of MES (morpholinoethelenediol sulfonic acid) buffer (pH 6), and As(III) was added to final concentration of 1.33 mM. The flasks were incubated at 30Â°C for 2 hour. Samples were removed after every 30 min, and arsenic concentration was determined as described earlier.
2.6. Spheroplast preparation and dosage of arsenite oxidase activity
Bacteria grown in the presence of As(III) (100 mg/liter) were pelleted, resuspended in 20 mM Tris-HCl, 0.1 mM phenylmethylsulfonyl fluoride 10 mM EDTA (pH 8.4) containing 20% sucrose, and treated with 0.5 mg/ml of lysozyme for 40 min at 25Â°C (Anderson et al., 1992). Arsenite oxidase activity was determined based on the transfer of reducing equivalents from arsenite to 2,4-dichlorophenolindophenol (DCIP). Reduction of DCIP (60 ÂµM) was monitored at 600 nm in the presence of 200 ÂµM sodium arsenite in 50 mM MES buffer, pH 6.0, at 25Â°C. Specific activity was defined as micromoles of DCIP reduced per minute per milligram of protein (Anderson et al., 1992).
2.7. Finding of aoxB gene
The PCR amplification of and arsenic marker genes was performed using bacterial genomic DNA as a template. Sequences of the PCR primers for arsenic marker gene were (Forward primer 5'GTCGGYTGYGGMTAYCAYGYYTA3' Reverse primer 5'YTCDGARTTGTAGGCYGGBCG3') as described by Inskeep et al. (2007). Degenerate primers used to amplify the arsenic marker genes were respectively designed especially for aoxA, or aoxB genes. The PCR programme include cycling program included 35 cycles of denaturation (94Â°C, 1 min), annealing (48Â°C, 1 min) and extension (72Â°C, 2 min). Initial denaturation (94Â°C, 2 min) and final extension (72Â°C, 5 min) were performed at the end of the 30 cycles. The amplified PCR products were purified using a Mini BEST DNA Fragment Purification kit (Takara, Japan). The presence of aoxB gene in the PCR product was confirmed by sequencing at Beijing Genomics Institutes, Beijing with an ABI Prism 3730 DNA analyzer (Applied Biosystems, USA).
2.8. Nucleotide sequence accession numbers. The DNA sequences described in this report have been submitted to GenBank under accession number JX448407 (16s rRNA gene) and JX657678 (aoxB gene).
2.9. Statistical analysis
Data were statistically analysed by analysis of variance (ANOVA) and the mean differences were compared by Tukey-Kramer Multiple Comparison Test at p <0.05. Three replicates were maintained for each treatment. All the analyses were performed using GraphPad Prism (v 4.03) software.
3.1. Isolation, identification, and characterization of XS3
The As(III) oxidizing bacteria were isolated by enrichment culture technique. Out of 12 different bacterial strains isolated from enrichment technique, only one strain, XS3 showed positive As(III) oxidation reaction by silver nitrate screening method. Other isolates showing negative As(III) oxidation reaction were discarded and strain -XS3 were further studied. Isolated strains were Gram-negative and short rods. XS3 was motile. XS3 were identified by sequencing 16S rRNA, and their phylogenetic analysis was performed. This strain belonged to Proteobacteria group and strain XS3 showed 98% (97% coverage) similarity to Pseudomonas thivervalensis strain McBPA3 (Genbank accession number JQ317814) In phylogenetic analysis (Figure 1) XS3, together with Pseudomonas thivervalensis. This strain showed high resistance towards As (III). Growth of XS3 was observed in presence of 1000 mg/l As(III). Utilization of different carbon sources by strains was tested and strain XS3 used citrate, lactate, and succinate. Gluconate and malate was used.
3.2. Growth and As(III) oxidation by strain XS3
Growth and As(III) oxidation ability was tested in SLP containing 100, 200, 400, 800, 1600, 3200, 6400 and 1000 mg/l of As(III). The growth of strain XS3 was not significantly affected by the presence of 800 mg/l (AsIII) but little affected at 3200 and 6400 mg/l of As(III) (Figure 2a). The growth of XS3 was increased significantly just after the inoculation and showed maximum growth at 24 hours. XS3 strain showed rapid oxidation of As(III) in SLP medium. Strain XS3 oxidized 200 mg/l of As(III) completely within 8 and 12 hour, respectively (Figure 2b), this able to oxidized efficiently up to 6400 mg/l of As(III) (Figure 3a&b). XS3 strains began As(III) oxidation at the start of exponential phase, and it was complete before the end of exponential phase, it was also observed that cell density continued to increase even after complete oxidation of As(III).
3.3. Induction of arsenite oxidase system
Arsenite oxidase system of XS3 was induced by arsenic, As(III). The As(III) oxidation was not observed when cells were grown without arsenic (Figure 4). However, oxidation was observed 40% for strain XS3 when cells were grown in presence of As(III).
3.4. Localization of arsenite oxidase
The presence of arsenite oxidase was tested in both fractions supernatant as well as biomass during the sphaeroplast preparation. Approximately 10% of total activity was found in released content from the periplasmic space. Thus the protein seems to be located in the membrane fraction showing 90% of the activity. Specific activity was calculated for arsenite oxidase from both supernatant as well as from biomass, we found that supernatants having 0.012 ÂµM protein/min and in biomass or pellate 0.1 ÂµM protein/min.
3.5. Arsenic marker genes
The aoxB sequences were amplified from strain XS3 arsenite oxidizers. The deduced gene sequence of aoxB from Pseudomonas thivervalenisis XS3 showed 94% (coverage 91%) identity to aoxB gene from Pseudomonas arsenooxidans (GenBank accession no. FN824370). Also showed similarity with uncultured organism clone VD-1d arsenite oxidase Mo-pterin subunit (aoxB) gene (GeneBank accession no. DQ380576 and DQ380577 showed 98% (coverage 91%) identity to aoxB gene (Figure 5). The primers mentioned in material and method section correspond to the aoxB genes. Isolated bacterial strain, XS3 was found to be positive for the (Mo-pterin subunit) aoxB arsenic marker genes. Phylogenetic relationships were obtained by comparing the aoxB gene sequences of the bacterial isolates with sequences of members of previously reported arsenic-resistant, As(III)-oxidizing, bacteria and closely related bacterial isolates. The aoxB gene sequences of XS3 showing close similarity with different previous reported genera (Figure 5). Previous reported sequences obtained from GeneBank.
As(III)-oxidizing bacteria can contribute to a natural attenuation of As pollution by decreasing its bioavailability and can help remove As from mine wastewaters through bioprocessing (Afkar et al., 2003; Ahmann et al., 1994). Many As(III) oxidizers have been isolated from various environments, especially mesophilic ecosystems (Anderson et al., 1992; Arai et al., 2003; Chowdhury 2004). They belong to more than 25 genera, mainly of the Proteobacteria phylum (Anderson et al., 1992; Messens et al., 2002; Nealson et al., 2002) and are related to organisms unable to oxidize As(III) based on 16S rRNA phylogeny.
In this research, we investigated the relevant As(III)-oxidizing bacteria that exists in highly arsenic-contaminated mine tailing area of Xinjiang, China. Present Isolate XS3 was able to oxidize at high concentration than any previous reported bacterial isolates. XS3 were able to transform arsenite up to 6400 mg/l concentration. Nearly full-length 16S rRNA gene sequences were used for bacterial identification which shows maximum similarity with Pseudomonas thivervalensis. Phylogenetically diverse arsenite-oxidising bacteria have been isolated from various aquatic and soil environments. It has been shown to be induced in the presence of arsenite. The biological oxidation of arsenite using bacteria is of particular interest for decontamination of arsenic-contaminated waste or ground water. Microbial oxidation of arsenite is a critical link in the global As cycle by converting more toxic arsenite into less mobile and less toxic arsenate species (Ehrlich, 2002).
Arsenite oxidation was catalyzed by a periplasmic arsenite oxidase. This enzyme contains two subunits encoded by the genes aoxA/aroB/asoB (small Fe-S Rieske subunit) and aoxB/aroA/asoA(large Mo-pterin subunit) respectively (Mukhopadhyay et al., 2002; Santini et al., 2004). Recently aoxB-like sequences have been widely found in different arsenic contaminated soil and water systems (Inskeep et al., 2007). Present result by XS3 also showed arsenite oxidase a membrane bound protein were observed in both supernatant as well as in biomass or pellate. Which support the bacteria to metabolize high concentration of arsenite. Arsenite oxidase is probably transported over the cytoplasmic membrane via the tat system and remains membrane attached by an N-terminal trans-membrane helix of the Rieske subunit as suggested by Lebrum et al. (2003).
Diverse primer sets have been successfully developed to specifically target the functional aoxB gene (Conrads et al., 2002; Frankenberger, 2002; Inskeep et al., 2002; Lievremont et al., 2003; Macy et al., 1996) encoding the large molybdenum-bearing catalytic subunit of As(III)- oxidase (EC 18.104.22.168), an enzyme of the dimethyl sulfoxide (DMSO) reductase family. Using cloning-sequencing approaches, the aoxB gene has proven to be a reliable molecular marker for diversity studies of the polyphyletic aerobic As(III) oxidizers in As-impacted soil and water systems (Inskeep et al., 2002; Lievremont et al., 2003).
Inskeep et al. (2007) reported that arsenite oxidase genes are widely present in different arsenite oxidizers and widespread in soil-water systems. We have enriched gold mine tailing sample with arsenite to isolate arsenite-oxidizing bacteria. To our knowledge, all of the cultured arsenite oxidizers obtained so far were isolated from arsenic-contaminated sites. Inskeep et al. (2007) detected aoxB-like sequences from arsenic-contaminated environments but not from gold mines sample indicating that arsenite oxidation is a major process in arsenic contaminated environments. The expression level of aoxB could probably be applied to monitor environmental arsenic-contaminated levels. Present studies also found the arsenite marker aoxB gene. This indicates that aoxB may be specific for most aerobic bacteria and use full for detecting arsenite oxidizing microorganism in the environment.
A phylogenetic analysis of the arsenite oxidizers based on the aoxB genes showed a similar phylogeny indicating genomic stability of the aoxB genes. The arsenite oxidizer displayed a maximal arsenite resistance level by XS3. Furthermore, strain containing the arsenite oxidase showed a higher arsenite resistance level. These results suggest that bacteria capable of arsenite oxidations using arsenite oxidase have an elevated arsenite resistance level (Figure 3&b).
Increased arsenic levels will result in new selective pressures for arsenic resistance, and an increased importance of examining microbial arsenic resistance in natural environments. Microbial metabolism undoubtedly exacerbates environmental arsenic problems (Frankenberger, 2002; Inskeep et al., 2002). Understanding the mechanisms may help minimize the impact. Present research provides valuable information of microbial species and gene responsible for arsenite oxidation and resistance, and increases knowledge of the distribution of the indigenous bacteria that may be stimulated for successful bioremediation of arsenic contamination.
Present research found highly efficient bacterial isolate XS3 for asenite oxidation which is not reported earlier. This study provides valuable source of microorganism (and genes) that may contribute to arsenic abnormality and may be useful in bioremediation of As(III).
This work was supported by Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences and National Natural Science Foundation of China(U1120302 and 21177127).