Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
236 Characterization of arsenic-resistant bacteria from the rhizosphere of arsenic hyperaccumulator Pteris vittata Anhui Huang, Max Teplitski, Bala Rathinasabapathi, and Lena Ma Abstract: Arsenic hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates that are hypothesized to solubilize arsenic and maintain a unique rhizosphere microbial community. Total heterotrophic counts on rich or defined media supplemented with up to 400 mmol/L of arsenate showed a diverse arsenate-resistant microbial community from the rhizosphere of P. vittata growing in arsenic-contaminated sites. Twelve bacterial isolates tolerating 400 mmol/L of arsenate in liquid culture were identified. Selected bacterial isolates belonging to different genera were tested for their resistance to osmotic and oxidative stresses. Results showed that growth was generally better under osmotic stress generated by arsenic than under that generated by NaCl or PEG 6000, demonstrating that arsenic detoxification metabolism also crossprotected bacterial isolates from arsenic-induced osmotic stress. After 32 h of growth, all arsenate at 1 mmol/L was reduced to arsenite by strains Naxibacter sp. AH4, Mesorhizobium sp. AH5, and Pseudomonas sp. AH21, but arsenite at 1 mmol/L remained unchanged. Sensitivity to hydrogen peroxide was similar to that in broad-host pathogen Salmonella enterica sv. Typhimurium wild type, except strain AH4. The results suggested that these arsenic-resistant bacteria are metabolically adapted to arsenic-induced osmotic or oxidative stresses in addition to the specific bacterial system to exclude cellular arsenic. Both these adaptations contribute to the high arsenic resistance in the bacterial isolates. Key words: arsenic-resistant bacteria, Pteris vittata, osmotic stress, oxidative stress, arsenate reduction. Résumé : La fougère Pteris vittata L. qui accumule de hauts niveaux d’arsenic, produit une grande quantité d’exsudat racinaire que l’on croit capable de solubilizer l’arsenic et de maintenir une communauté microbienne unique dans la rhizosphère. Les comptes totaux d’hétérotrophes sur du milieu riche ou défini supplémenté à l’arsenate à des concentrations allant jusqu’à 400 mmol/L ont montré la présence d’une communauté microbienne résistante à l’arsenate dans la rhizosphère de P. vittata cultivée sur des sites contaminés à l’arsenic. Douze isolats bactériens tolérant 400 mmol/L d’arsenate en culture liquide ont été identifiés. Des isolats bactériens sélectionnés appartenant à différents genres ont été testés quant à leur résistance à des stress osmotiques et oxydatifs. Les résultats ont montré l’existence d’une meilleure croissance en général sous un stress osmotique généré par l’arsenic comparativement au chlorure de sodium ou au PEG 6000, démontrant que le métabolisme de détoxication de l’arsenic protège les isolats bactériens du stress osmotique causé par l’arsenic de façon croisée. Après 32 h de croissance, tout l’arsenate à 1 mmol/L était réduit en arsénite par les souches Naxibacter sp. AH4, Mesorhizobium sp. AH5 et Pseudomonas sp. AH21, mais l’arsenite à 1 mmol/L demeurait inchangé. Leur sensibilité au peroxyde d’hydrogène était similaire à celle du pathogène à large spectre Salmonella enterica sv. Thypimurium sauvage, à l’exception de la souche AH4. Les résultats ont suggéré que ces bactéries résistantes à l’arsenic sont métaboliquement adaptées au stress osmotique induit par l’arsenic ou au stress oxydatif, en plus de posséder un système bactérien d’exclusion d’arsenic cellulaire spécifique. Ces deux adaptations contribuent à la haute résistance à l’arsenic des isolats bactériens. Mots-clés : bactéries résistantes à l’arsenic, Pteris vittata, stress osmotique, stress oxydatif, réduction de l’arsenate. [Traduit par la Rédaction] Introduction Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) tolerates up to 1500 mg/kg of arsenic in soil and rapidly accumulates up to 2.3% arsenic in its aboveground biomass from contaminated soils (Ma et al. 2001). Pteris vittata produces more root exudates than non-hyperaccumulator fern Nephrolepis exaltata; the root exudates are hypothe- sized to help P. vittata mobilize arsenic directly from soils (Tu et al. 2004). Besides directly affecting arsenic mobilization from soils, root exudates also help sustain a rhizosphere microbial community, which indirectly mobilizes arsenic from soil. In a symbiosis between plant roots and rhizosphere microbes, plants produce carbon compounds, while microbes Received 27 April 2009. Revision received 30 November 2009. Accepted 11 January 2010. Published on the NRC Research Press Web site at cjm.nrc.ca on 18 March 2010. A. Huang, M. Teplitski, and L. Ma.1 Soil and Water Science Department, University of Florida, Gainesville, FL 32601, USA. B. Rathinasabapathi. Horticultural Science Department, University of Florida, Gainesville, FL 32601, USA. 1Corresponding author (e-mail: [email protected]). Can. J. Microbiol. 56: 236–246 (2010) doi:10.1139/W10-005 Published by NRC Research Press Huang et al. help the plant to maintain nutrient recycling, provide resistance to microbial diseases, and tolerate toxic compounds (Morgan et al. 2005). Microbial activities in the rhizosphere of P. vittata impact the fate of arsenic by changing soil pH, root exudation, and redox potential, thus playing a key role in controlling arsenic bioavailability in soils (Fitz and Wenzel 2002). Besides interacting with plant roots, bacteria also play an important role in arsenic biogeochemistry in the environment. They affect arsenic reduction and oxidation, methylation and demethylation, and sorption and desorption in soils. As a result, bacteria have developed different detoxification strategies to withstand the growth restriction under arsenic stress. On one hand, bacterial ability to tolerate osmotic and oxidative stress contributes to their arsenic resistance. Metabolism under hyperosmotic conditions or in a low nutrient environment could cross-protect cells from other stresses such as oxidative burst, heavy metal stress, and sodium hypochlorite (Pichereau et al. 2000). Similar to other heavy metals, arsenic causes oxidative stress by inducing reactive oxygen species (ROS). Therefore, it is not surprising to see the correlation between arsenic resistance and hydrogen peroxide (H2O2) resistance (Liu et al. 2001). On the other hand, arsenic-resistant bacteria usually have a specific genetic system, which is directly involved in arsenic transformation and sequestration. One example is the reduction–detoxification mechanism, which has been found in bacteria isolated from arsenic-contaminated soils and mine tailings (Jones et al. 2000; Macur et al. 2001). The functional genes are encoded by the arr operon in dissimilatory arsenic-reducing prokaryotes (DARP) (Rosen 2002) or by the ars operon in arsenite-specific expulsion prokaryotes (ASEP) (Kuai et al. 2001). The fact that the soil where arsenic hyperaccumulation was first found in P. vittata has been contaminated for approximately 60 years (Komar 1999), coupled with the ability of P. vittata to solubilize arsenic in the rhizosphere, provides a good opportunity to discover microbes highly tolerant to arsenic. The objectives of this study were to isolate and characterize arsenic-resistant bacteria from arseniccontaminated soils where P. vittata naturally grows. The ability of the bacterial isolates to tolerate osmotic and oxidative stresses induced by different sources was studied, and functional genes in the specific genetic system were investigated. Materials and methods Bacterial isolation and enumeration by total heterotrophic counting Soil (bulk and rhizosphere) and plant samples were collected from 2 arsenic-contaminated sites where P. vittata grows naturally in north central Florida in April 2007. The first site (CCA site) was contaminated by chromated copper arsenate (CCA), which was used for pressure-treating lumber from 1951 to 1962 (Komar et al. 1998). The second site (RES site) was a residential site where CCA-treated timber was used for stairs and decks. Rhizosphere soil, defined as the soil attached to the roots, was removed from the roots by gentle shaking. Bulk soil without plant influence was collected from the soil near the sampling plant at the same site. Soil samples were kept at 4 8C for 20 days before bacterial 237 isolation. For arsenic analysis, soil and plant samples were air dried (22 8C), mixed thoroughly, and digested by HNO3–H2O2 (US Environmental Protection Agency Method 3051) on a heating block in triplicates (Environmental Express, Ventura, Calif.). Arsenic concentrations in the solutions were analyzed by a graphite furnace atomic absorption spectrophotometer (240Z, Varian, Walnut Creek, Calif.). To select arsenic-resistant bacteria, 5 soil samples from the CCA and RES sites with different arsenic concentrations were used. The CCA soils included 2 bulk soils (92.8 and 167 mg/kg of arsenic) and 1 rhizosphere soil (80.0 mg/kg of arsenic), and the RES soils included one bulk soil (12.9 mg/kg of arsenic) and one rhizosphere soil (28.2 mg/kg of arsenic). Soil samples (0.3 g) were suspended in 10 mL of sterilized water and vortexed vigorously. Soil suspensions (2, 20, and 200 mL) were plated onto 2 agarose media: modified tryptone – yeast extract – glucose (TYEG, 1/10 strength) or the National Botanical Research Institute’s phosphate growth medium (NBRIP). The 1/10 strength TYEG medium contained 1 g/L of tryptone, 0.3 g/L of yeast extract, 0.5 g/L of glucose, and 1% agarose (TYEG medium used in this study was all at 1/10 strength). The NBRIP agarose medium contained 5 g/L of glucose, 5 g/L of MgCl 26H 2O, 0.25 g/L of MgSO47H2O, 0.2 g/L of KCl, 0.1 g/L of (NH4)2SO4, 10 mg/L of FeSO4, 14 mg/L of nitric acid, and 1% agarose, which was spiked with inorganic or organic phosphorus sources (5 g of phosphate rock, Ca 10(OH)2(PO4)6(F, Cl), or sodium phytic acid, Na 12C 6H 6O 24P6). After 2 days, the 3 media with the highest number of single colonies among 3 volumes’ worth of inocula were inoculated to plates containing 6 different levels of arsenate (10, 50, 100, 200, 300, and 400 mmol/L of sodium arsenate). The number of surviving bacterial colonies was counted after 2 days of growth. Single colonies from plates containing 400 mmol/L of arsenate were subcultured 3 times to obtain a pure culture. Since arsenic availability in solid media might be limited, bacteria isolated from solid plates containing 400 mmol/L of arsenic were inoculated into TYEG liquid medium containing 400 mmol/L of arsenic to screen the most arsenictolerant bacteria. All bacterial incubations were conducted at room temperature (22 8C). Identification of arsenic-resistant bacteria The 12 most arsenic-resistant bacteria were identified by sequencing and analyzing the 16S rRNA gene. Bacterial genomic DNA was extracted using the standard phenol– chloroform method followed by ethanol precipitation. The 16S rDNA fragments were PCR amplified using Taq polymerase and primers 8F 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1489R 5’-TACCTTGTTACGACTTCA-3’ (Bruneel et al. 2006). The PCR products were cloned using the TOPO TA cloning kit (Invitrogen Inc., Carlsbad, Calif.) and sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) Sequencing Facility at the University of Florida (Gainseville, Fla.). Bacteria were identified based on 16S rDNA sequences. Two-sequence comparisons among the 12 isolates were analyzed by BLAST (Altschul et al. 1990). Published by NRC Research Press 238 Bacterial growth under osmotic stress Eight bacterial isolates tolerating 400 mmol/L of sodium arsenate in liquid culture and belonging to 8 different genera according to 16S rRNA gene sequences (Naxibacter sp. AH4, Rhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, and Caryophanon sp. AH28) were used in this study. TYEG medium was used for this study. The experiment was conducted under –1.5 MPa of osmotic stress using 3 different sources: 176 mmol/ L of sodium arsenate, 400 mmol/L of NaCl, and 26% polyethylene glycol 6000 (PEG 6000) (Sosa et al. 2005), with the TYEG medium as a control. Bacteria were grown at room temperature (22 8C) with constant shaking (150 r/ min). Bacterial growth was measured at 0, 5, 16, 25, 38, and 44 h after inoculation by using a spectrophotometer (Shimadzu BioSpec-Mini; Shimadzu Biotechn Inc., Columbia, Md.) at 600 nm (1 cm light-path length). Bacterial growth inhibition under oxidative stress The same 8 bacterial isolates were tested for their resistance to oxidative stress by measuring inhibition area for H2O2 on TYEG agar. Salmonella enterica sv. Typhimurium GS014 oxyR::Tn10 and Salmonella enterica sv. Typhimurium 14028 wild type were used as controls. For the assays, 50 mL aliquots of bacterial culture grown overnight were plated in TYEG agar medium, and bacterial isolates were allowed to grow for 4 h. A sterile paper disc (6 mm in diameter) was placed inside each plate, to which 25 mL of 3% (v/v) H2O2 was applied. Plates were incubated at 22 8C, and the diameters of the inhibition zones were measured after 24 and 48 h. The experiments were carried out in triplicate. Can. J. Microbiol. Vol. 56, 2010 Sigma-Aldrich, St. Louis, Mo.). Total soluble proteins were extracted by vortexing with glass beads and centrifuging at 4 8C. Protein concentration was determined using the BioRad protein assay (Peterson 1979) (Bio-Rad Laboratories, Hercules, Calif.). The arsenate reductase assay solution contained 150 mL of protein extract, 1 mL of reaction buffer, 10 mmol/L of sodium arsenate, 0.5 mmol/L of NADPH, 1 mmol/L of glutathione, 2 U of yeast glutathione reductase, and 0.02 mmol/L of Escherichia coli glutaredoxin 2. Enzyme activity was determined by measuring the absorption at 340 nm (Gladysheva et al. 1994; Shi et al. 1999). Bacterial arsenate reductase gene The primers used for arsC gene amplification were 5’-GCATTCTTTCCGAAGCCATGTTCAA-3’ (forward) and 5’-AGCTCACGCTTGAGCTGGTCGCGAT-3’ (reverse), which were designed based on Pseudomonas aeruginosa PAO1 arsC gene. Pseudomonas aeruginosa PAO1 was used as a positive control, and the PCR product from PAO1 was confirmed by sequencing before being used used as a probe for Southern hybridization. The PCR conditions used were 94 8C for 7 min and 29 cycles at 94 8C for 1 min; 56.8 8C for 1 min; 72 8C for 1 min; and a final extension at 72 8C for 10 min, with 0.2 mmol/L of primers. The Southern blot method of Sambrook and Russell (2001) and the user manual of the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Indianapolis, Ind.) were followed. Results Arsenic transformation by arsenic-resistant bacteria Arsenic transformation by 3 arsenic-resistant bacterial isolates (Naxibacter sp. AH4, Mesorhizobium sp. AH5, and Pseudomonas sp. AH21) and a non-arsenic resistant bacterium, Sinorhizobium meliloti MG32, were analyzed. The experiment included a control without bacteria and 3 replicates per treatment. The growth medium contained either 1 mmol/L of arsenite (AsIII) or arsenate (AsV). All bacterial cultures had the same initial cell density (OD600 = 0.1). Cell cultures were grown at room temperature with constant shaking at 150 r/min. Shake cultures were sampled after 4, 8, 16, and 32 h. Total arsenic was analyzed by a graphite furnace atomic absorption spectrophotometer. Arsenic speciation was performed using an arsenic speciation cartridge (Metal Soft Center, Highland Park, N.J.) (Meng et al. 2001). Diversity of the arsenic-resistant microbial community Arsenic-resistant bacteria were isolated from 2 arseniccontaminated soils by growth in both rich medium, TYEG, and defined medium, NBRIP. For both sites, the rhizosphere of P. vittata sustained a substantially higher number of arsenic-resistant bacteria compared to that found in the bulk soils with similar arsenic concentrations (Tables 1 and 2). Taking the CCA site for example, 43% of the colonies from the rhizosphere soil grew in TYEG containing 400 mmol/L of arsenate, while the numbers in the 2 bulk soils from the same site were 17% and 3.4%, respectively (Table 1). In the RES site, the percentage of surviving isolates in TYEG plates containing 400 mmol/L of arsenate was 13% and 3.1% for rhizosphere soil and bulk soil, respectively. In total, 53 isolates, designated AH1–AH53, were obtained from the plates containing 400 mmol/L of arsenate. Isolates AH1– AH4 were also phosphate-solubilizing bacteria isolated in the defined NBRIP medium. Arsenate reductase assay Cell culture of arsenic-resistant Pseudomonas sp. AH45 was grown in 50 mL of TYEG medium overnight. To induce the synthesis of proteins regulated by arsenic, 0.1 mmol/L of sodium arsenate was added to the medium, which was incubated for 3 h. The medium was then incubated in a shaker (200 r/min) for another 4 h before the cells were harvested by centrifugation. Cell pellets were resuspended in reaction buffer (10 mmol/L of Tris (pH 7.5), 1 mmol/L of Na2EDTA, 1 mmol/L of MgCl2, and 1 mmol/L of DTT) (Anderson and Cook 2004) with 0.1 mmol/L of proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, Twelve most arsenic-resistant bacterial isolates Chemoheterotrophic bacteria tolerating 400 mmol/L of arsenate were isolated from rhizosphere soils. Because TYEG is a nutrient-rich medium and NBRIP is a defined medium, bacteria recovered through growth in NBRIP were able to grow in TYEG medium. Therefore, for better comparison in the growth study, TYEG medium was used for all isolates. Considering that arsenic availability in agarose plates is lower than that in liquid culture, 53 isolates were further screened in TYEG liquid culture containing 400 mmol/L of arsenate. Those able to grow were further separated and identified based on 16S rRNA gene sequence analyses using Published by NRC Research Press Huang et al. 239 Table 1. Percentage of arsenate-resistant bacterial colonies recovered in tryptone – yeast extract – glucose (TYEG) medium inoculated with bacteria isolated from 2 arsenic-contaminated soils.a Arsenate concentration in TYEG medium (mmol/L) Soilb CCA B1 CCA B2 CCA R3 RES B4 RES R5 Soil arsenic (mg/kg) 92.5 167 80.0 12.9 28.0 10 62.3 40.9 68.1 15.9 78.7 50 46.8 37.5 66.0 9.50 76.0 100 42.9 36.4 57.4 4.80 74.7 200 40.3 21.6 46.8 3.10 72.0 300 36.4 15.9 42.5 3.10 69.3 400 16.9 3.40 42.5 3.10 13.3 a Percentage of surviving colonies as a proportion of c.a. 80 single colonies in each soil tested. b CCA site, chromated copper arsenate contaminated site; RES site, residential contaminated site; B, bulk soil; R, rhizosphere soil. Table 2. Percentage of bacterial colonies surviving in the National Botanical Research Institute’s phosphate growth medium (NBRIP, containing phosphate rock as phosphorus source) under different arsenate concentrations.a Arsenate concentration in NBRIP medium (mmol/L) Soilb CCA B1 CCA B2 CCA R3 RES B4 RES R5 Soil arsenic (mg/kg) 92.5 167 80.0 12.9 28.0 10 87.2 98.9 54.3 0 53.7 50 38.5 73.3 0 0 0 100 25.6 14.4 20.0 0 24.1 200 15.4 4.40 20.0 0 0 300 5.1 0 0 0 0 400 0 0 0 0 0 a Percentage of surviving colonies as a proportion of c.a. 80 single colonies in each soil tested. b CCA site, chromated copper arsenate contaminated site; RES site, residential contaminated site; B, bulk soil; R, rhizosphere soil. Table 3. The 12 most arsenic-resistant bacteria identified from 2 arsenic-contaminated soils. Bacterial IDa AH4 AH5 AH6 AH10 AH21 AH22 AH23 AH25 AH28 AH34 AH43 AH45 Soil arsenic (mg/kg) 28.2 92.8 92.8 92.8 80.0 80.0 80.0 80.0 80.0 80.0 28.2 28.2 Soil typeb R B B B R R R R R R R R Phylogenetic neighborc, GenBank accession No. Naxibacter sp., AM774589 Mesorhizobium sp., AM181745 Methylobacterium sp., AM910536 Enterobacter sp., Z96079 Pseudomonas sp., AF368755 Bacillus sp., FJ188297 Acinetobacter sp., AM945567 Pseudomonas sp., EF178450 Caryophanon sp., AF385535 Pseudomonas sp., EF178450 Pseudomonas sp., AF368755 Pseudomonas sp., EF178450 a All bacteria were from the CCA site (chromated copper arsenate contaminated site) except for AH4, AH43, and AH45, which were from the RES site (residential contaminated site). b R, rhizosphere soil; B, bulk soil. c 16S rRNA gene; reference is selected from published data with similar cellular morphologies. Published by NRC Research Press 240 BLAST (Table 3). The 12 most arsenic-resistant soil isolates were tentatively identified as Naxibacter sp. AH4, Mesorhizobium sp. AH5, Methylobacterium sp. AH6, Enterobacter sp. AH10, Pseudomonas sp. AH21, Bacillus sp. AH22, Acinetobacter sp. AH23, Pseudomonas sp. AH25, Caryophanon sp. AH28, Pseudomonas sp. AH34, Pseudomonas sp. AH43, and Pseudomonas sp. AH45. Their most probable phylogenetic neighbors, which were selected based on published data and similar colony morphologies, showed that 2 groups of isolates (AH21 and AH43; AH25, AH34, and AH45) shared the same most probable neighbors. The results were consistent with comparative analyses of 2 sequences at a time via BLAST based on 600 informatic nucleotides of the 16S rRNA genes among the 12 isolates (data not shown). The two-sequence comparisons via BLAST also showed that, while 2 groups of isolates (AH21 and AH43; AH25, AH34, and AH45) had 99% similarity; other isolates all had similarity below 97%. Bacterial growth under osmotic stress To test whether the observed resistance to arsenate simply resulted from a high resistance to osmotic stress, growth of arsenic-resistant bacteria was tested under various osmotic stresses. The growth characteristics of the 8 most arsenicresistant bacteria were studied under –1.5 MPa osmotic stress induced by 400 mmol/L of NaCl, 26% (m/v) of PEG 6000 or 176 mmol/L of sodium arsenate. A laboratory strain, P. fluorescens CHA0, which was pretested for its sensitivity to osmotic stress, was used as a control. Except for the control CHA0, all bacteria tolerated –1.5 MPa osmotic stress (Fig. 1). Figure 1 shows the data for bacterial isolates AH4, AH10, and AH23. Bacterial isolates AH5, AH6, AH21, AH22, and AH28 were comparable with AH10 (data not shown). Compared to the growth curve in TYEG, all 8 bacterial isolates tested had a longer lag phase under osmotic stress generated by PEG 6000. All 8 isolates grew better under NaCl-induced osmotic stress than under that induced by PEG 6000. At the end of the experiment, all bacterial isolates grew better under arsenic than under NaCl or PEG 6000, except for AH23. The control P. fluorescens CHA0 tolerated –1.5 MPa of osmotic stress, but was unable to grow in the presence of arsenic. To compare bacterial ability to tolerate different stresses, a resistance index (RI) was calculated. The RI was defined as the ratio of the exponential growth rate in the medium with stress to that in the control medium. The closer RI was to 1, the smaller the arsenate toxicity was. The RI for arsenateinduced stress for the 8 isolates was the highest for AH28 (0.94) and the lowest for AH23 (0.20) (Fig. 2). The RI index showed that, among the 3 sources of stress, bacterial growth rate in arsenate was the highest, with a mean RI of 0.62 for the 8 strains studied, followed by NaCl with mean of 0.37, and PEG6000 was the lowest with mean of 0.29. AH23 was unusual among the arsenic-resistant bacteria in that it exhibited greater tolerance to NaCl than to arsenate (Fig. 2). Bacterial growth inhibition under oxidative stress Arsenate also causes oxidative stress in bacteria. To evaluate bacterial oxidative stress tolerance, H2O2 inhibition of the 8 isolates was compared to inhibition of Salmonella enterica sv. Typhimurium GS014 oxyR::Tn10 and Salmonella Can. J. Microbiol. Vol. 56, 2010 enterica sv. Typhimurium 14028 wild type. These controls were chosen because they were well characterized for their responses to oxidative stress. The bacteria were tested for antioxidative stress by measuring the H2O2 inhibition zone relative to the controls. Under the experimental conditions, the largest inhibition diameters occurred for AH4, with 47 mm after 24 h and 43 mm after 48 h (Fig. 3). The smallest inhibition was observed for AH28, with 22 mm after 24 h and 21 mm after 48 h. The results showed that, except for AH4, the arsenic-resistant bacteria were as resistant as Salmonella enterica sv. Typhimurium 14028 wild type to H2O2. AH4 was as sensitive as the oxyR mutant (Fig. 3) (p < 0.001), suggesting that the arsenic and NaCl tolerances of AH4 are not related to its H2O2 tolerance. In vitro arsenic transformation by arsenic-resistant bacteria Three arsenic-resistant bacteria, Naxibacter sp. AH4, Mesorhizobium sp. AH5, and Pseudomonas sp. AH21, were tested for their ability to transform arsenic during 32 h of growth in TYEG medium, which was spiked with 1 mmol/L of arsenate or arsenite (375 mg for 5 mL of cell culture). A non-arsenic resistant strain, Sinorhizobium sp. MG32, which was pretested for its resistance to different arsenic species, was used as a control. Speciation of arsenic in TYEG medium without bacterial inoculation confirmed that both arsenate and arsenite were stable in shaking TYEG medium. While there was neither oxidation nor reduction in the medium with Sinorhizobium sp. MG32, all 3 arsenic-resistant bacterial isolates reduced arsenate to arsenite efficiently in 32 h (Fig. 4a), with the halflife of arsenate being 22 h for AH4, 18 h for AH5, and 12 h for AH21. Figure 4b shows the final OD at 600 nm after 32 h of growth in the presence of arsenic. The growth of control bacterium Sinorhizobium sp. MG32 was inhibited by 1 mmol/L of arsenite, whereas all 3 arsenic-resistant bacteria tolerated both arsenate and arsenite. Bacterial arsenate reductase and the functional gene To test the presence of arsenate reductase in the bacterial isolates, protein extract from AH45 was tested for activity in vitro. The coupled assay experiment tested the activity of glutaredoxin-dependent arsenate reductase (glutaredoxin 2 from E. coli), with NADPH/arsenate being the electron donor/acceptor. Compared to the control without protein extracts or glutaredoxin, arsenate reduction by E. coli glutaredoxin 2 was observed, with absorption decreasing at a rate of 0.0012 ABS/min (Fig. 5). Enzymatic reduction by soluble protein from Pseudomonas sp. AH45 was detected from the decrease of NADPH absorption at 340 nm, with absorption decreasing at rate of 0.0045 ABS/min, translating to a specific activity of 3.58 nmolmin–1mg–1 (Gladysheva et al. 1994). Although a portion of the arsC gene was successfully amplified using PCR, with the genomic DNA of the positive control as the template, and the partial arsC gene confirmed by sequencing, there was no positive PCR product amplified from arsenic-resistant bacteria by the primers designed based on the arsC gene of P. aeruginosa PAO1. Southern hybridization with the arsC probe (partial gene sequence, Published by NRC Research Press Huang et al. 241 Fig. 1. Growth curve of Pseudomonas fluorescens CHA0 (A) and 3 arsenic-resistant bacteria, AH4, AH10, and AH23 (B, C, and D), in tryptone – yeast extract – glucose (TYEG) medium under osmotic stress (–1.5 MPa) generated by 400 mmol/L of NaCl, 176 mmol/L of sodium arsenate, or 26% (m/v) of PEG 6000. Data are means and standard errors of 3 replicates. Standard errors were not shown when they were smaller than the depicted points. A 0 -0.5 -1 -1.5 -2 -2.5 -3 0 -0.5 -1 -1.5 -2 TYEG NaCl PEG As -2.5 -3 0 5 10 15 20 25 30 35 40 0 0.5 C Growth (log10 OD600 ) 0.5 Growth (log10 OD600 ) B 0.5 Growth (log10 OD600 ) Growth (log10 OD600 ) 0.5 0 -0.5 -1 -1.5 -2 -2.5 5 10 15 20 25 10 15 20 25 Time (h) 30 35 30 35 40 D 0 -0.5 -1 -1.5 -2 -2.5 -3 -3 0 5 10 15 20 25 Time (h) 30 35 0 40 5 40 Fig. 2. Resistance index (RI) of 8 arsenic-resistant bacteria (AH4–AH28) based on growth at –1.5 MPa of osmotic stress. RI was defined as the ratio of the growth rate in medium with –1.5 MPa of osmotic stress to that in the control medium. Pseudomonas fluorescens CHA0 was a laboratory control strain. Data are means and standard errors of 3 replicates. 1.2 CHA0 AH4 AH5 AH6 0.8 AH10 AH21 0.6 AH22 AH23 Resistance Index 1 0.4 AH28 0.2 0 PEG NaCl As Stress 408 nt) from PAO1 also showed a negative result (data not shown). GenBank accession No. of the 16S rRNA gene sequences of the 12 isolates are as follows: AH4: FJ621305; AH5: FJ621306; AH6: FJ621307; AH10: FJ621308; AH21: FJ621309; AH22: FJ621310; AH23: FJ621311; AH25: FJ621311; AH28: FJ621313; AH34: FJ621314; AH43: FJ621315; AH45: FJ621316. Discussion Previous studies have reported that microbial arsenic resistance is associated with P. vittata. For example, the bacterial isolate AsRB1 from the phyllosphere exhibited resistance to arsenate, arsenite, and antimony and reduced arsenate to arsenite (Rathinasabapathi et al. 2006). Plant growth and arsenic uptake were increased by either inoculation with whole arbuscular mycorrhizal fungal community (Al Agely et al. 2005) or with specific fungus Glomusmosseae or Gigasporamargarit (Trotta et al. 2006). The fungi that were studied all tolerated the spiked arsenic (£100 mg/kg) in the soil. However, so far there has been no study focused on the rhizosphere bacterial community associated with P. vittata. Published by NRC Research Press 242 Can. J. Microbiol. Vol. 56, 2010 Fig. 3. Diameter of the growth inhibition zone of 8 arsenic-resistant bacteria (AH4–AH28), Salmonella enterica sv. Typhimurium oxyR::Tn10 (GS014), and S. enterica sv. Typhimurium wild type (WT) in a glass disc containing 25 mL of 3% hydrogen peroxide. The y-axis represents the diameter of the inhibition area on Petri dishes. Data are means and standard errors of 3 replicates. 50 24 h 48 h Inhibition(mm) 40 30 20 10 0 AH4 AH5 AH6 AH10 AH21 AH22 AH23 AH28 GS014 WT Strain Fig. 4. Arsenic transformation by arsenic-resistant bacteria AH4, AH5, and AH21 and control strain Sinorhizobium sp. MG32 during 32 h of growth in tryptone – yeast extract – glucose (TYEG) medium spiked with 375 mg of arsenate (As(V)) or arsenite (As(III)). (A) Arsenic speciation in the medium. The y-axis shows the amount of the transformed arsenic (reduction on the left axis and oxidation on the right axis). Solid and broken lines denote the amount of arsenite and arsenate, respectively, that was transformed by bacterial isolates. R, reduction test; O, oxidation test. (B) Growth of AH4, AH5, AH21, and M32 after 32 h in TYEG. Medium spiked with 375 mg of arsenate (solid column) or arsenite (open column). Points and columns are means and standard errors of 3 replicates. 400 400 300 300 200 200 100 100 0 0 -100 -100 4 8 R AH4 R Control O MG32 12 16 20 Time(h) 24 R AH5 O AH4 O Control B 28 32 R AH21 O AH5 Reduction R MG32 O AH21 Oxidation 1.2 OD600 AsV oxidation (µg) AsIII reduction (µg) A 0.8 0.4 0 AH4 AH5 Strain AH21 MG32 Diversity of arsenic-resistant microbial community Under the selective pressure that existed in the contaminated sites for several decades, the microbial community was selected for its ability to overcome growth restrictions through arsenic detoxification. The proportion of arsenicresistant bacteria was presented as a percentage of the number of bacteria colonies tested (Table 1), which minimized the effects of nutrition and soil heterogeneity. The fact that a higher percentage of arsenic-resistant bacteria was from the rhizosphere soils than from the bulk soils indicated that, during plant arsenic accumulation, a greater amount of bioavailable arsenic was mobilized from soil that selected for the arsenic-resistant microbial community in the rhizosphere. Phosphorus plays a major role in arsenic detoxification by P. vittata. Under arsenic stress, the tendency of P. vittata to take up more phosphorus has been attributed to arsenicinduced phosphorus deficiency (Tu and Ma 2003). Phosphorus availability in phytic acid or phosphate rock was much lower than that provided in the TYEG medium. The fact that the percentage of phosphorus-solubilizing bacteria surviving at a high arsenic concentration was much lower than the percentage of isolates surviving in TYEG (Table 2) implies that bacterial ability to solubilize phosphorus was not critical to its ability to tolerate arsenic. Alternatively, the bacteria failed to obtain sufficient phosphorus to help detoxify the arsenic. Moreover, by supplying bacteria with a high arsenic concentration in the NBRIP medium, phosphorus-solubilizing bacteria might have taken up more arsenic through the nonspecific phosphorus transporter and thus suffered more arsenic toxicity. A previous study described a similar level of arsenic resistance in Corynebacterium glutamicum, which tolerated 500 mmol/L of arsenate on tryptic soy agar plates (Ordóñez et al. 2005). However, arsenic bioavailability in solid medium is reduced not only because of low arsenic activity, but also because of the small bacterial surface area that directly contacts the arsenic. This study used 1/10 strength TYEG medium to simulate the low nutrient environment in soil, and more importantly, minimized the potential of a nutrient-induced reduction in arsenic availability. Therefore, bacterial arsenic resistance was tested in a liquid culture. The difference in arsenic bioavailability between solid and liquid cultures was obvious from our data. Among the 53 isolates surviving on solid 1% agarose plates containing 400 mmol/L of arsenate, only 12 (23%) grew in liquid culture with the same arsenic concentration. Therefore, those isolates represent the most arsenic-resistant bacteria. The 12 strains were identified by sequencing the 16S rRNA gene. Two-sequence comparisons via BLAST showed that 8 of the strains had a similarity <97% (data not shown), which was the borderline similarity level to define a species (Paster et al. 1991). The high similarity (99%) among isolates AH25, AH34, and AH45 suggested that the 3 isolates might be from the same species or strain. Bacterial growth under osmotic stress In soils under –1.5 MPa of osmotic stress (the permanent wilting point for many plants), the water film thickness surrounding soil matrices is estimated to be ~10 H2O molecules (Halverson and Firestone 2000). Under the same osmotic Published by NRC Research Press Huang et al. 243 Fig. 5. Arsenate reductase assay of arsenic-resistant bacterium AH45, where enzyme activity was detected by measuring NADPH absorption at 340 nm. The total protein concentration in the reaction was 101 mg/mL. Data are means and standard errors of 3 replicates. 1.5 ABS (340 nm) 1.45 1.4 1.35 1.3 1.25 Background Grx no protein AH45 1.2 0 1 2 3 4 stress in bacterial medium, bacteria are expected to desiccate because of reduced water availability. This study showed that arsenic-resistant bacteria grew in the presence of 400 mmol/L of arsenate in 1/10 strength TYEG medium, a medium simulating the low nutrient conditions in soil. However, most of the isolates were unable to grow under similar ionic strength induced by 2.4 mol/L of NaCl (data not shown). Instead of using a lethal dose of NaCl, this growth experiment used a lower concentration (400 mmol/L), which the 8 arsenic-resistant isolates were able to tolerate. The osmotic stresses induced by NaCl and PEG 6000 were both hyperosmotic, representing low water stress with and without electrolytes. The reason for using 2 different osmotic stresses was that, in some cases, a decrease in water potential was due primarily to reduction in water activity (e.g., PEG 6000) rather than to an increased concentration of permeating salts (e.g., NaCl). High molecular weight PEG 6000 was too large to penetrate cell walls, lowering medium water potential in the way that a dry soil would. Bacteria tolerate osmotic stress through accumulation of cytoplasmic solutes (Csonka 1989). However, ionic and nonionic stresses such as NaCl and PEG 6000 had different effects on compatible solute uptake in bacteria, which can affect the maintenance of the electrolyte balance in the cytoplasm, thus affecting bacterial growth (Molenaar et al. 1993). Therefore, NaCl stress might be expected to have effects more similar to those of arsenate stress than those of PEG 6000 stress. Heavy metal detoxification by bacteria can result from sorption to biomass, sequestration, and intracellular precipitation. A previous study showed that arsenate interfered with the regulation of Rhizobium sp. VMA301 cell wall biosynthesis, which might result from arsenic binding to cell walls (Mandal et al. 2008). Moreover, phytochelatins, heavy metal binding peptides, are present in many prokaryotes, including cyanobacterium Nostoc (Anabaena) sp. PCC 7120, Prochlorococcus marinus strain MIT9313 (BX572098), and Anabaena variabilis ATCC29413 (Hirata et al. 2005). Similar to plants hyperaccumulating heavy metals, phytochelatins in bacteria would confer a high degree of heavy metal tolerance. Transformation–immobilization is not only affected by bacterial functional enzymes, but also by the reduction– oxidation potential in the environment, two processes that are greatly influenced by microbial communities (Gadd 2004). Therefore, arsenic detoxification can be the byproduct 5 6 Time (min) 7 8 9 10 of normal metabolism, making it difficult to search for the functional genes. From this angle, cellular physiological studies are important in understanding mechanisms of arsenic resistance. Compared to the bacterial growth curve observed for TYEG, all bacteria had a longer lag phase under osmotic stress (Fig. 1). Arsenic-resistant bacteria generally grew better under NaCl-induced osmotic stress than under osmotic stress induced by PEG 6000 (Fig. 1). The presence of ions in the media might help to maintain membrane potential during the accumulation of osmoprotectants. Under osmotic stress induced by PEG 6000, a low availability of cations and anions might have dissipated the cell membrane. With the same amount of inoculation, the initial absorption at 600 nm in bacterial culture with PEG 6000 was smaller than in TYEG medium with either NaCl or arsenate for all strains. This suggested that bacterial cells underwent plasmolysis because of the osmotic pressure (low water activity outside cellular membrane). Most bacteria except AH23 grew better with arsenic than with NaCl or PEG6000, indicating the existence of crossprotection between arsenic toxicity and arsenate-generated osmotic stress. The control P. fluorescens CHA0 tolerated –1.5 MPa of osmotic stress, but was unable to grow in the presence of arsenic (Fig. 1). Therefore, cellular growth might be under the influence of bacterial ability to detoxify arsenic and bacterial resistance to low water potential induced by permeating solute. In fact, multiple tolerances frequently occur in bacteria living under osmotic stress or starvation conditions as a result of global reprogramming of gene expression. For example, Hartke et al. (1998) demonstrated that under complete starvation conditions induced in tap water, Enterococcus faecalis cells became more tolerant to heat, acid, and sodium hypochlorite stresses and were significantly more resistant to UV (245 nm) irradiation. A recent study of gene profile changes responding to pH change in Shigella flexneri by whole-genome microarrays differentiated the expression of 307 genes. The genes included global regulators such as the s factors and specific genes that increased acid production and energy generation (Cheng et al. 2007). Northern blot comparison of Listeria monocytogenes mRNA between growth in neutral and alkaline environments revealed that expression of ~60 sB-regulated genes was significantly increased, and the bacterial strain became more resistant to Published by NRC Research Press 244 subsequent exposure to ethanol, alkaline, or osmotic stress (Giotis et al. 2008). Those studies discerned that global regulons conferred cross-protection against multiple stresses such as oxidative burst, osmotic stress, as well as heavy metal toxicity. The higher RI induced by NaCl than that induced by PEG 6000 (Fig. 2) confirmed the different effects of ionic and nonionic osmotic stresses on bacterial cell growth. Figure 2 also shows that most bacteria had a higher tolerance to arsenic than to NaCl, supporting the idea that arsenic detoxification metabolism likely cross-protected bacteria from arsenic-induced ionic osmotic stress. The higher resistance of AH23 to NaCl than to arsenic suggested that it might have a unique arsenic detoxification mechanism, and its tolerance to osmotic stress could contribute more to the arsenic resistance of AH23. The closer the arsenic RI is to 1, the lower the arsenic toxicity. Though RI is affected by the medium and growth conditions, it can still provide a relative comparison of bacterial ability to detoxify arsenic among isolates in different studies. For example, compared to the control, the growth of isolate AsRB1 from the phyllosphere of P. vittata was about 20% in Luria–Bertani medium containing 10 mmol/L of arsenate (Rathinasabapathi et al. 2006). Both the arsenic concentration and the growth percentage were much lower than in our study, in which the lowest RI for AH23 was even higher than 0.20 (Fig. 3). Bacterial growth inhibition under oxidative stress The oxyR regulon controls oxidative burst by increasing ROS scavenging activities and limiting H2O2 generation in the respiratory chain (González-Flecha and Demple 2000). Under exposure to heavy metals, glutathione and sulfhydryl groups in proteins are depleted after complexing with metals, which leads to production of ROS such as superoxide ion, H2O2, as well as hydroxyl radicals (Stohs and Bagchi 1995). With Tn10 inserted in the oxyR gene, the mutant GS014 is very sensitive to oxidative stress. Bacterial defenses against oxidative burst include antioxidant enzymes such as superoxide dismutase and catalase, DNA repair systems following damage by ROS, scavenging substrates, and competition with phagocytes for molecular oxygen. Some of these defenses are regulated by global regulon oxyR (Hassett and Cohen 1989). The fact that the arsenic-resistant bacteria were as resistant as Salmonella enterica sv. Typhimurium wild type was to H2O2 was consistent with a previous report that oxyR protected bacteria from arsenic toxicity (Sukchawalit et al. 2005). Bacterial ability to scavenge ROS during an oxidative burst might contribute to their high arsenic resistance. The result was supported by a previous study on P. aeruginosa, which showed that mutants lacking the arsenite membrane pump ArsB, superoxide dismutase, catabolite repression control protein, or glutathione reductase were all more sensitive than the wild type to arsenite (Parvatiyar et al. 2005). Bacterium Naxibacter sp. AH4 was as sensitive as Salmonella enterica sv. Typhimurium GS014 to oxidative stress in the inhibition test (Fig. 3). As a chemical analog of phosphate, arsenate is taken up by phosphate transporters in bacteria (Rosen 2002). With such high sensitivity to H2O2, AH4 needed to sequester arsenic by rapid efflux–immobilization Can. J. Microbiol. Vol. 56, 2010 before arsenic-induced oxidative burst occurred inside cells and prevented arsenic uptake. Therefore, lack of downstream oxidative detoxification ability in AH4 suggested a different arsenic detoxification mechanism compared to that in other isolates, probably a mechanism that involved suppressing the nonselective uptake system. Bacterium AH4 was also the only one able to grow in the presence of 2.4 mol/L of NaCl (data not shown). Further study is needed to unveil the specific genes that confer the unique detoxification ability in this strain. Bacterial arsenate reductase and functional gene DARP and ASEP are 2 groups of known arsenate-reducing bacteria (Plant et al. 2003). The transformation experiment showed a high efficiency of arsenate reduction by the bacterial isolates, while no oxidation was observed (Fig. 4). The experiment was carried out in rich medium under aerobic conditions; therefore, those isolates tested might belong to ASEP, and the functional genes were not constitutively expressed according to the growth curve (Fig. 1). Under the ASEP model, bacteria may take up arsenate through a phosphate transporter, reduce it to arsenite using a specific enzyme with the help of glutaredoxin, thioredoxin, or ferredoxin, and extrude the arsenite by membrane pumps (Rosen 2002). After being normalized on the basis of cell growth, the RI indicates the comprehensive effects of the bacterial ability to reduce and extrude arsenic in this model. The high RI of bacterial isolates Mesorhizobium sp. AH5, Enterobacter sp. AH10, and Bacillus sp. AH28 reflects their enhanced ability to detoxify arsenic. The chemical transformation results and enzymatic assay proved the existence of a specific genetic system for transforming and detoxifying arsenic by arsenic-resistant bacteria that belonged to DARP or ASEP. Although a positive control was sequenced and confirmed in the experiment cloning the arsenate reductase gene in ASEP, there was no positive PCR product amplified from 12 arsenic-resistant bacteria by the primers designed based on the arsC gene sequence of P. aeruginosa PAO1. The negative results were confirmed by several degenerate primers designed based on reported ars sequences that were phylogenetically close to our isolates. A genomic DNA Southern blot using the arsC gene from P. aeruginosa PAO1 as a probe also showed negative results (data not shown), suggesting the probable sequence divergence from arsC known in P. aeruginosa PAO1. Phylogenetic analysis of bacterial and archaeal arsC gene sequences showed that arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms; in addition, the corresponding arsC gene sequences had little or no sequence similarity and led to various homologies of the corresponding arsC protein (60%–90%) (Jackson and Dugas 2003; Branco et al. 2008). Therefore, our results suggested that either novel functional arsenate reductase genes or low similarity between the arsenic-resistant bacteria and the positive control resulted in difficulties in identifying the target gene. Previous studies showed that certain DARP species were more sensitive to arsenic than others. For example, haloalkaliphile Bacillus selenitireducens grew well at 10 mmol/L of arsenate, while Sulfurospirillum species was only able to grow at 5 mmol/L of arsenate. This could be explained by a Published by NRC Research Press Huang et al. generally weak arsenic-resistant system in Sulfurospirillum species, in which an increase in the pH of the medium impeded the exit of arsenite from cells (Ahmann et al. 1994; Switzer Blum et al. 1998). In our current study, while AH4 was sensitive to oxidative stress (Fig. 3), it tolerated high levels of arsenic. AH23 had the highest NaCl resistance but the lowest arsenic resistance (Fig. 2). In summary, we isolated and characterized a group of arsenic-resistant and (or) phosphorus-solubilizing bacteria from the rhizosphere of the arsenic hyperaccumulator P. vittata. Twelve isolates tolerated 400 mmol/L of arsenate in liquid culture, the highest arsenic resistance reported. Most studies on arsenic-resistant bacteria have focused on specific arsenic-resistant mechanisms, such as functional genes involved in detoxification mechanisms through transformation and sequestration of arsenic species; however, little information is available on global metabolic adaptations such as the osmotic stress between membranes when arsenic is stored and (or) accumulated, oxidative stress generated during exposure to arsenic, and the impacts of those stresses on cell growth. In this study, for the first time, we examined crosstolerances between the arsenic resistance of bacterial isolates and their tolerance to stress induced by NaCl, PEG 6000, and H2O2. While specific functional enzymes controlled the uptake, reduction, and extrudation of arsenic, global metabolism counteracted arsenic-induced stresses, such as oxidative burst or osmotic stress. Experimental results suggested that bacterial ability to resist arsenic was due to bacterial efficiency in transforming and sequestering arsenic, scavenging oxidative burst, and counteracting different osmotic stresses. In addition, the bacterial isolates can potentially be used for bioleaching to remove arsenic and phosphorus from soils. These bacteria can also be used to increase the phosphorus availability in agricultural soils as well as in arseniccontaminated soils to improve plants’ phosphorus nutrition. References Ahmann, D., Roberts, A.L., Krumholz, L.R., and Morel, F.M. 1994. Microbe grows by reducing arsenic. Nature (Lond.), 371(6500): 750. doi:10.1038/371750a0. PMID:7935832. Al Agely, A., Sylvia, D.M., and Ma, L.Q. 2005. Mycorrhizae increase arsenic uptake by the hyperaccumulator Chinese brake fern (Pteris vittata L.). J. Environ. Qual. 34(6): 2181–2186. doi:10.2134/jeq2004.0411. PMID:16275719. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215(3): 403–410. PMID:2231712. Anderson, C.R., and Cook, G.M. 2004. Isolation and characterization of arsenate-reducing bacteria from arsenic-contaminated sites in New Zealand. Curr. Microbiol. 48(5): 341–347. doi:10. 1007/s00284-003-4205-3. PMID:15060729. Branco, R., Chung, A.P., and Morais, P.V. 2008. Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T. BMC Microbiol. 8(1): 95. doi:10.1186/1471-2180-895. PMID:18554386. Bruneel, O., Duran, R., Casiot, C., Elbaz-Poulichet, F., and Personné, J.C. 2006. Diversity of microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France. Appl. Environ. Microbiol. 72(1): 551–556. doi:10.1128/AEM.72.1.551-556. 2006. PMID:16391091. Cheng, F., Wang, J., Peng, J., Yang, J., Fu, H., Zhang, X., et al. 245 2007. Gene expression profiling of the pH response in Shigella flexneri 2a. FEMS Microbiol. Lett. 270(1): 12–20. doi:10.1111/j. 1574-6968.2007.00647.x. PMID:17286558. Csonka, L.N. 1989. Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53: 121–147. Fitz, W.J., and Wenzel, W.W. 2002. Arsenic transformations in the soil–rhizosphere–plant system: fundamentals and potential application to phytoremediation. J. Biotechnol. 99(3): 259–278. doi:10.1016/S0168-1656(02)00218-3. PMID:12385714. Gadd, G.M. 2004. Microbial influence on metal mobility and application for bioremediation. Geoderma, 122(2-4): 109–119. doi:10.1016/j.geoderma.2004.01.002. Giotis, E.S., Julotok, M., Wilkinson, B.J., Blair, I.S., and McDowell, D.A. 2008. Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and crossprotection against subsequent ethanol and osmotic stress. J. Food Prot. 71(7): 1481–1485. PMID:18680951. Gladysheva, T.B., Oden, K.L., and Rosen, B.P. 1994. Properties of the arsenate reductase of plasmid R773. Biochemistry, 33(23): 7288–7293. doi:10.1021/bi00189a033. PMID:8003492. González-Flecha, B., and Demple, B. 2000. Genetic responses to free radicals. Homeostasis and gene control. Ann. N.Y. Acad. Sci. 899: 69–87. PMID:10863530. Halverson, L.J., and Firestone, M.K. 2000. Differential effects of permeating and nonpermeating solutes on the fatty acid composition of Pseudomonas putida. Appl. Environ. Microbiol. 66(6): 2414–2421. doi:10.1128/AEM.66.6.2414-2421.2000. PMID: 10831419. Hartke, A., Giard, J.C., Laplace, J.M., and Auffray, Y. 1998. Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl. Environ. Microbiol. 64(11): 4238–4245. PMID:9797271. Hassett, D.J., and Cohen, M.S. 1989. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3(14): 2574–2582. PMID:2556311. Hirata, K., Tsuji, N., and Miyamoto, K. 2005. Biosynthetic regulation of phytochelatins, heavy metal-binding peptides. J. Biosci. Bioeng. 100(6): 593–599. doi:10.1263/jbb.100.593. PMID: 16473766. Jackson, C.R., and Dugas, S.L. 2003. Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase. BMC Evol. Biol. 3(1): 18. doi:10.1186/1471-2148-3-18. PMID:12877744. Jones, C.A., Langner, H.W., Anderson, K., McDermott, T.R., and Inskeep, W.P. 2000. Rates of microbially mediated arsenate reduction and solubilization. Soil Sci. Soc. Am. J. 64: 600–608. Komar, K.M. 1999. Phytoremediation of arsenic contaminated soils: plant identification and uptake enhancement. M.S. thesis, University of Florida, Gainesville, Fla. Komar, K., Ma, L.Q., Rockwood, D., and Syed, A. 1998. Identification of arsenic tolerant and hyperaccumulating plants from arsenic contaminated soils in Florida. Agron. Abstr. 343. Kuai, L., Nair, A.A., and Polz, M.F. 2001. Rapid and simple method for the most-probable-number estimation of arsenicreducing bacteria. Appl. Environ. Microbiol. 67(7): 3168–3173. doi:10.1128/AEM.67.7.3168-3173.2001. PMID:11425737. Liu, S.X., Athar, M., Lippai, I., Waldren, C., and Hei, T.K. 2001. Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc. Natl. Acad. Sci. U.S.A. 98(4): 1643– 1648. doi:10.1073/pnas.031482998. PMID:11172004. Ma, L.Q., Komar, K.M., Tu, C., Zhang, W., Cai, Y., and Kennelley, E.D. 2001. A fern that hyperaccumulates arsenic. Published by NRC Research Press 246 Nature (Lond.), 409(6820): 579. doi:10.1038/35054664. PMID: 11214308. Macur, R.E., Wheeler, J.T., McDermott, T.R., and Inskeep, W.P. 2001. Microbial populations associated with the reduction and enhanced mobilization of arsenic in mine tailings. Environ. Sci. Technol. 35(18): 3676–3682. doi:10.1021/es0105461. PMID: 11783644. Mandal, S.M., Pati, B.R., Das, A.K., and Ghosh, A.K. 2008. Characterization of a symbiotically effective Rhizobium resistant to arsenic: isolated from the root nodules of Vigna mungo (L.) Hepper grown in an arsenic-contaminated field. J. Gen. Appl. Microbiol. 54(2): 93–99. doi:10.2323/jgam.54.93. PMID: 18497483. Meng, X., Korfiatis, G.P., Jing, C., and Christodoulatos, C. 2001. Redox transformations of arsenic and iron in water treatment sludge during aging and TCLP extraction. Environ. Sci. Technol. 35(17): 3476–3481. doi:10.1021/es010645e. PMID: 11563649. Molenaar, D., Hagting, A., Alkema, H., Driessen, A.J., and Konings, W.N. 1993. Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis. J. Bacteriol. 175(17): 5438–5444. PMID:8366030. Morgan, J.A.W., Bending, G.D., and White, P.J. 2005. Biological costs and benefits to plant-microbe interactions in the rhizosphere. J. Exp. Bot. 56(417): 1729–1739. doi:10.1093/jxb/ eri205. PMID:15911554. Ordóñez, E., Letek, M., Valbuena, N., Gil, J.A., and Mateos, L.M. 2005. Analysis of genes involved in arsenic resistance in Corynebacterium glutamicum ATCC 13032. Appl. Environ. Microbiol. 71(10): 6206–6215. doi:10.1128/AEM.71.10.6206-6215. 2005. PMID:16204540. Parvatiyar, K., Alsabbagh, E.M., Ochsner, U.A., Stegemeyer, M.A., Smulian, A.G., Hwang, S.H., et al. 2005. Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite. J. Bacteriol. 187(14): 4853–4864. doi:10. 1128/JB.187.14.4853-4864.2005. PMID:15995200. Paster, B.J., Dewhirst, F.E., Weisburg, W.G., Tordoff, L.A., Fraser, G.J., Hespell, R.B., et al. 1991. Phylogenetic analysis of the spirochetes. J. Bacteriol. 173(19): 6101–6109. PMID:1917844. Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100(2): 201–220. doi:10.1016/0003-2697(79)90222-7. PMID:393128. Pichereau, V., Hartke, A., and Auffray, Y. 2000. Starvation and osmotic stress induced multiresistances. Influence of extracellular compounds. Int. J. Food Microbiol. 55(1-3): 19–25. doi:10. 1016/S0168-1605(00)00208-7. PMID:10791712. Plant, J.A., Kinniburgh, D.G., Smedley, P.L., Fordyce, F.M., Klinck, B.A., Heinrich, D.H., and Karl, K.T. 2003. Arsenic and Can. J. Microbiol. Vol. 56, 2010 selenium. Treatise Geochem. 9: 17–66. doi:10.1016/B0-08043751-6/09047-2. Rathinasabapathi, B., Raman, S.B., Kertulis, G., and Ma, L. 2006. Arsenic-resistant proteobacterium from the phyllosphere of arsenic-hyperaccumulating fern (Pteris vittata L.) reduces arsenate to arsenite. Can. J. Microbiol. 52(7): 695–700. doi:10. 1139/W06-017. PMID:16917527. Rosen, B.P. 2002. Biochemistry of arsenic detoxification. FEBS Lett. 529(1): 86–92. doi:10.1016/S0014-5793(02)03186-1. PMID:12354618. Sambrook, J., and Russell, D. 2001. Molecular cloning. 3rd ed. Vol. 1. Cold Spring Harbor Laboratory Press, Plainview, N.Y. Shi, J., Vlamis-Gardikas, A., Aslund, F., Holmgren, A., and Rosen, B.P. 1999. Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction. J. Biol. Chem. 274(51): 36039–36042. doi:10.1074/jbc.274.51.36039. PMID: 10593884. Sosa, L., Llanes, A., Reinoso, H., Reginato, M., and Luna, V. 2005. Osmotic and specific ion effects on the germination of Prosopis strombulifera. Ann. Bot. (Lond.), 96(2): 261–267. doi:10.1093/ aob/mci173. Stohs, S.J., and Bagchi, D. 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18(2): 321–336. doi:10.1016/0891-5849(94)00159-H. PMID:7744317. Sukchawalit, R., Prapagdee, B., Charoenlap, N., Vattanaviboon, P., and Mongkolsuk, S. 2005. Protection of Xanthomonas against arsenic toxicity involves the peroxide-sensing transcription regulator OxyR. Res. Microbiol. 156(1): 30–34. doi:10.1016/j. resmic.2004.07.005. PMID:15636745. Switzer Blum, J., Burns Bindi, A., Buzzelli, J., Stolz, J.F., and Oremland, R.S. 1998. Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Arch. Microbiol. 171(1): 19–30. doi:10.1007/ s002030050673. PMID:9871015. Trotta, A., Falaschi, P., Cornara, L., Minganti, V., Fusconi, A., Drava, G., and Berta, G. 2006. Arbuscular mycorrhizae increase the arsenic translocation factor in the As hyperaccumulating fern Pteris vittata L. Chemosphere, 65(1): 74–81. doi:10.1016/j. chemosphere.2006.02.048. PMID:16603227. Tu, C., and Ma, L.Q. 2003. Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L. Plant Soil, 249(2): 373–382. doi:10.1023/A:1022837217092. Tu, S.X., Ma, L., and Luongo, T. 2004. Root exudates and arsenic accumulation in arsenic hyperaccumulating Pteris vittata and non-hyperaccumulating Nephrolepis exaltata. Plant Soil, 258(1): 9–19. doi:10.1023/B:PLSO.0000016499.95722.16. Published by NRC Research Press