ledbetter et al. 2007 aem 1 biogenic mineral production by a novel

Ledbetter et al. 2007 AEM

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Biogenic mineral production by a novel arsenic-metabolizing thermophilic bacterium from 1 the Alvord Basin, Oregon, USA 2 3 Rhesa N. Ledbetter1, Stephanie A. Connon1, Andrew L. Neal2, Alice Dohnalkova3, and Timothy 4 S. Magnuson1* 5 6 1 Department of Biological Sciences, Idaho State University, Pocatello, Idaho 83209 7 2 Savannah River Ecology Laboratory and Department of Crop and Soil Sciences, University of 8 Georgia, Athens, Georgia 30602 9 3 Pacific Northwest National Laboratory, Richland, Washington 99352 10 * Corresponding Author 11 12 Revised Manuscript: For re-submission to Applied and Environmental Microbiology, 6-23-07. 13 14 Short Title: Novel As-reducing bacterium from the Alvord Basin 15 16 Corresponding Author Contact Information: 17 Dr. Timothy S. Magnuson 18 Department of Biological Sciences 19 P.O. Box 8007 20 Idaho State University 21 Pocatello, ID 83209 22 Phone: 208-282-5014 23 FAX: 208-282-4570 24 Email: [email protected] 25

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00371-07 AEM Accepts, published online ahead of print on 13 July 2007

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ABSTRACT 1

The Alvord Basin in southeast Oregon, USA contains a variety of hydrothermal features 2

which have never been microbiologically characterized. Sampling of Murky Pot (61flC, pH 7.1) 3

led to the isolation of a novel arsenic-metabolizing organism (YeAs) which produces an arsenic 4

sulfide mineral known as く-realgar, a mineral that has not previously been observed as a product 5

of bacterial arsenic metabolism. YeAs was grown on a freshwater medium and utilized a variety 6

of organic substrates, particularly carbohydrates and organic acids. The temperature range for 7

growth was 37-75°C (optimum, 55°C), and the pH range for growth was 6.0-8.0 (optimum, pH 8

7.0-7.5). No growth was observed when YeAs was grown under aerobic conditions. The 9

doubling time when grown with yeast extract and As(V) was 0.71 hours. Microscopic 10

examination revealed Gram-indeterminate, non-spore forming, non-motile, rod-shaped cells, 0.1-11

0.2 µm x 3-10 µm in dimension. Arsenic sulfide mineralization of cell walls and extracellular 12

arsenic sulfide particulate deposition were observed with electron microscopy and elemental 13

analysis. 16S rRNA gene analysis placed YeAs in the order Clostridiales and indicated the 14

organism is most closely related to Caloramator and Thermobrachium species. The G+C content 15

was 35%. YeAs showed no detectable respiratory arsenate reductase but did display significant 16

detoxification arsenate reductase activity. The phylogenetic, physiological, and morphological 17

characteristics of YeAs demonstrate that it is an anaerobic, moderately thermophilic, arsenic-18

reducing bacterium. This organism and its associated metabolism could have major implications 19

in the search for innovative methods for arsenic waste management and in the search for novel 20

biogenic mineral signatures. 21

Keywords: く-realgar, arsenic reduction, mineral biosynthesis, anaerobic metabolism 22

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INTRODUCTION 1

Microorganisms play an important role in the biogeochemical cycling of many elements 2

(34) including arsenic. Arsenic is relatively abundant in soils and natural waters, and although it 3

is primarily a consequence of anthropogenic sources, it also occurs naturally in many minerals 4

(19, 26) and in groundwaters in contact with geologic formations containing high levels of 5

arsenic. In arsenic-laden environments, the compound can exist in three different oxidation states 6

which include As(V) (arsenate), As(III) (arsenite), and As(-III) (arsine) (10). Microorganisms are 7

capable of transforming the various forms of arsenic using mechanisms for oxidizing As(III) or 8

reducing As(V) (19). These mechanisms, although commonly a mode of detoxification, can also 9

be a source of energy generation through the use of As(V) as a terminal electron acceptor (19) or 10

As(III) as an electron donor. Depending of the type of mechanism utilized, these processes 11

require distinct sets of genes/enzymes to perform the redox reaction (33). A total of twenty-four 12

prokaryotes, including members of the Crenarchaeota, Aquificae, Chrysiogenes, Deferribacteres, 13

low G+C Gram positive bacteria, Halanaerobacter, and Gamma-, Delta-, and 14

Epsilonproteobacteria, have been observed to utilize arsenic as a terminal electron acceptor (28, 15

35). Irrespective of the exact method of metabolism, the number and variety of arsenic-utilizing 16

organisms isolated from the environment clearly show that they play an important role in the 17

transformation and geochemical cycling of arsenic. 18

Murky Pot, located in the Alvord basin and part of the Mickey hydrothermal system, was 19

initially examined by determination of bacterial diversity using contemporary molecular 20

methods. These studies revealed the presence of a variety of 16S rRNA sequences, the most 21

abundant being those related to the Deltaproteobacteria and Acidobacteria (17). This paper 22

describes the isolation, cultivation, and characterization of an arsenic-metabolizing organism 23

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(YeAs) from Murky Pot hot spring. Although arsenic metabolism is common among 1

microorganisms, YeAs seems to be distinct in that it produces a novel biogenic mineral, く-2

realgar. Neither the mineral nor the associated metabolism has been observed among the 3

organisms most closely related phylogenetically to YeAs. 4

MATERIALS AND METHODS 5

Field site description. At the time of sampling, the temperature of Murky Pot (Figure 1) was 6

61°C and the pH was 7.1. The aqueous geochemistry of Murky Pot revealed significant levels of 7

arsenic (1.0 mg/L or about 15µM) as well as substantial levels of sodium, borate, and carbonate. 8

Enrichment, isolation, and cultivation. A growth medium prepared anoxically from 0.22 µm 9

filtered Murky Pot hot spring water was inoculated in the field with suspensions of 10

sediment/water from Murky Pot and supplemented with 0.5% (w/v) yeast extract and 2.5 mM 11

sodium arsenate (Na2HAsO4). Tubes were transported back to the laboratory in thermos bottles 12

filled with hot spring water at 55˚C and then transferred to incubators at the desired temperature 13

of 55°C. When growth was evident as judged by microscopy and appearance of a yellow 14

precipitate, cultures were transferred to a synthetic freshwater medium Orex-4 (26) with 0.5% 15

(w/v) yeast extract and 2.5 mM As(V). Orex-4 medium contained the following salts (g/L): 0.14 16

g of KH2PO4, 0.25 g NH4Cl, 0.5 g KCl, 1.0 g NaCl, 0.62 g MgCl2. The base medium was 17

amended with (per liter): 1.0 ml Wolfe’s mineral solution (38), 1.0 ml Wolfe’s vitamin stock 18

solution (38), 20 ml of 2.5% (w/v) cysteine solution, 20 ml 9.5% (w/v) bicarbonate solution, 20 19

ml 0.75% (w/v) CaCl2 solution. The Hungate technique was used throughout, and culture 20

headspaces contained an atmosphere of N2/CO2 (80:20) (22). One subculture, designated YeAs, 21

was propagated for further study. In order to obtain single colonies of YeAs, samples of 22

enrichment cultures were used as inoculum for Orex-4 solid medium containing 1.5% high-23

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melting agarose. Solid medium was prepared in anaerobic glass bottles, and the Hungate 1

technique was employed to create an anoxic atmosphere. Following sterilization, the anaerobic 2

bottles were inoculated and allowed to solidify. Yellow colonies appeared after 5 days of 3

incubation, and these colonies were picked and transferred to fresh solid medium. This procedure 4

was repeated two more times, after which the culture YeAs was maintained in liquid freshwater 5

medium with 0.5% (w/v) yeast extract and 2.5 mM As(V) and incubated at 55˚C unless 6

otherwise stated. 7

For some growth experiments, cysteine was eliminated from the medium to prevent 8

interference of the arsenic sulfide mineral precipitation. All other components and concentrations 9

(including the yeast extract and As(V)) remained the same. Even with the removal of cysteine, 10

trace amounts of sulfur were still present in the medium from the addition of the vitamin and 11

mineral solutions; however, mineral production was significantly reduced. 12

Determination of growth parameters. Cell growth was measured using direct cell counts. YeAs 13

was grown in Orex-4 and Orex-4 (-cys) supplemented with yeast extract and As(V) or in Orex-4 14

with yeast extract only. Subsamples were taken at time zero and every three hours following 15

until the cells were in stationary phase. At each time point, 1 µl of each sample was stained with 16

400 µl of 4’-6-diamidino-2-phenylidole (DAPI) stock solution (2.5 µg/ml) for 25 minutes and 17

then vacuum filtrated onto 0.22 µm black polycarbonate membranes (Millipore, Billerica, MA). 18

The membranes were then mounted onto glass slides with a drop of immersion oil. Four fields 19

were counted for each sample under a total magnification of 1000X using a Leica DMRB 20

microscope fitted with a DAPI filter. 21

To determine the temperature range for growth, YeAs was inoculated into Orex-4 (-cys) 22

containing yeast extract and As(V) and incubated at temperatures ranging from 37˚C to 75˚C at 23

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pH25˚C 6.8. The pH range was tested from pH55˚C 4-9. All tubes were incubated at 55°C. The pH 1

was tested prior to inoculation and also during late log phase to ensure proper buffering. YeAs 2

was also tested for its ability to grow aerobically on Orex-4 and Orex-4 (-cys) supplemented with 3

yeast extract and As(V). While growing in the presence of oxygen, all tubes were incubated in a 4

tissue culture rotator (Lab-Line Cel-Gro, Fisher Scientific, Pittsburgh, PA) at 30 rpm. For 5

temperature, pH, and oxygen tolerance experiments, culture turbidity was determined by 6

measuring the optical density (600 nm) until the cells were in stationary phase. All growth 7

parameter experiments were done in duplicate. 8

Substrate utilization. The substrates utilized by YeAs were assessed using Biolog Anaerobic 9

MicroPlates (Hayward, CA) (4, 5). The MicroPlates test the ability of a microorganism to utilize 10

or oxidize an array of carbon sources under anaerobic conditions using an artificial tetrazolium 11

colorimetric electron acceptor. The plates were inoculated with YeAs cells in triplicate and wells 12

exhibiting a strong color change due to the reduction of the tetrazolium dye when compared to 13

the negative control were selected as positive for substrate utilization. 14

Microscopy and morphologic characterization. The Gram type was tested using the Gram stain 15

(12), KOH test (7), and peptidoglycan isolation for cell wall analysis. The Schaeffer-Fulton 16

endospore stain (31) was used to determine if spores were present in a YeAs culture grown for 17

24 hours in Orex-4 with yeast extract and As(V) and in a culture incubated for 2 weeks under the 18

same conditions. Motility was determined using 0.4% and 0.7% semi-solid Orex-4 and Orex-4 (-19

cys) deeps supplemented with yeast extract and As(V). Deeps were inoculated with YeAs cells 20

and incubated for 24 hours at 55flC. 21

Two drops of a 24 hour liquid culture of YeAs grown in Orex-4 with yeast extract and 22

As(V) were placed on a microscope slide with a cover slip and then observed using Nomarski 23

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differential interference contrast microscopy (1). Specimens were examined using a Leica 1

DMRB microscope equipped with SPOT imaging software (Diagnostic Instruments, Sterling 2

Heights, MI). 3

Transmission and scanning electron microscopy (TEM, SEM) were performed at the 4

Environmental Molecular Sciences Laboratory (EMSL) at the Pacific Northwest National 5

Laboratory. YeAs cells grown for 24 hours in Orex-4 with yeast extract and As(V) were used for 6

the microscopy, and EM samples were prepared anaerobically in a glovebag. Copper grids 7

coated with a formvar support film and sputtered with carbon (Electron Microscopy Sciences, 8

Hatfield, PA) were used for the whole mounts TEM imaging. A drop of cellular suspension was 9

applied to a grid and gently blotted on a filter paper. Imaging and analyses were performed 10

employing a JEOL 2010 High resolution TEM operating at 200kV. Elemental analysis was 11

performed by energy dispersive spectroscopy (Oxford, ISIS) coupled to the TEM. Images were 12

collected digitally and analyzed using the Gatan Digital Micrograph software (Gatan, Pleasanton, 13

CA). Samples for SEM were fixed in 2.5% glutaraldehyde, gradually dehydrated in ethanol 14

series, and applied on a carbon tape. They were processed by critical point drying, sputter-coated 15

with carbon, and imaged with a LEO 942 SEM at 2kV. 16

Raman spectroscopy. Precipitate samples for Raman spectroscopy were prepared under a 95% 17

N2: 5% H2 atmosphere and where necessary under 785 nm illumination (to prevent 18

photoalteration of the sample). Precipitates were removed from YeAs culture tubes with a 19

syringe and needle and collected by centrifugation at 5000g for 15 minutes. The collected 20

precipitates were immediately transferred to an NMR tube, and the tube was sealed with a plastic 21

cap under anaerobic conditions before being transferred to the spectrometer. To prevent exposure 22

to light, all tubes were transferred wrapped in aluminum foil and placed in the spectrometer in a 23

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darkened laboratory. Spectra were obtained using a FT-Raman 960 spectrometer (Nicolet, 1

Thermo Electron Corp.) employing 1064 nm excitation from a Nd:YVO4 laser and a liquid-N2 2

cooled Ge detector. Spectra were collected at room temperature with 800 mW laser power at the 3

sample and at a spectral resolution of 1 cm-1. For photo-transformation studies, samples were 4

illuminated for 12 hours using a 900 mW ultra-violet light source (R-52 Mineralight™, Ultra-5

violet Products Inc., San Gabriel, CA) or placed in direct sunlight for 2 weeks. 6

く-realgar production byYeAs and phylogenetically related bacteria. Thermobrachium celere 7

and Caloramator coolhaasii were tested for their ability to produce く-realgar while cultured 8

under YeAs standard conditions (Orex-4 with yeast extract and As(V)). They were also tested 9

using their published growth media supplemented with 0.05% cysteine, 0.5% yeast extract, and 10

2.5 mM As(V) (11, 30). 11

G+C content and 16S rRNA gene analysis. The G+C content of YeAs was determined by 12

HPLC at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (20). 13

Genomic DNA was isolated from YeAs using the Qiagen DNeasy Tissue Kit (Valencia, CA), 14

and the 16S rRNA gene was amplified using PCR with eubacterial primers 8F (5’- AGA GTT 15

TGA TCM TGG CTC AG- 3’) and 1492R (5’- GGY TAC CTT GTT ACG ACT T- 3’). PCR 16

products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). 17

Sequencing was performed using an ABI Prism 377 DNA sequencer (Applied Biosystems, 18

Foster City, CA) with ABI Prism Big Dye terminator cycle sequencing reagent and the following 19

primers: 8F, 1492 R, and 338F (5’-ACI CCT ACG GGI GGC WGC-3’). Phylogenetic analysis 20

of the 16S rRNA gene was performed using ARB software (18). A neighbor-joining tree was 21

inferred using the Jukes and Cantor model to estimate evolutionary distances. Gaps and missing 22

sequence were masked out leaving 740 aligned characters in the analysis. Three Clostridium 23

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species were used as an outgroup. Percent similarities were determined from unmasked 1

sequences in ARB. 2

Enzyme assays for respiratory and detoxification arsenate reductases. Cell extracts of YeAs 3

were prepared by homogenization of cells harvested from a culture grown on Orex-4 (-cys) with 4

yeast extract and As(V) and were used for the following experiments. In order to identify the 5

enzyme responsible for arsenate reduction in YeAs, three approaches were used. These included 6

a spectrophotometric assay for respiratory arsenate reductase (Arr), zymography for the detection 7

of Arr, and a spectrophotometric assay for detoxification arsenate reductase (Ars). Arr was 8

assayed spectrophotometrically using the reduced viologen method (24). An in-gel zymography 9

approach was also used to identify Arr activity. Thermus AO3C was used as a positive control in 10

this experiment as it has been shown to respire on arsenate (Connon et al., submitted). Non-11

denaturing polyacrylamide gel electrophoresis (ND-PAGE) was employed to resolve the 12

respiratory arsenate reductase enzyme complex in an active state. To visualize activity, the 13

respiratory arsenate reductase activity stain was used and adopted from the respiratory nitrate 14

reductase enzyme activity stain as published by Vallejos (37). The only change in the procedure 15

was the addition of 1 g sodium arsenate in substitution for 1 g potassium nitrate. Negative 16

control gels (no arsenate added) were ran to ensure visualized bands were due to arsenate 17

reductase activity. Ars was assayed using an NADPH/glutathione assay (23). All 18

spectrophotometric assays and activity stains were performed at 55ºC. 19

RESULTS 20

Culture characteristics and morphology. 21

Copious amounts of yellow precipitate formed within 24 hours when the cells were 22

grown on Orex-4 with yeast extract and As(V) (Figure 2A), and microscopic examination 23

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revealed that the cells were straight to slightly curved rods (Figure 2B). The cells primarily 1

occurred singly, and no endospores were observed by microscopy in a 24 hour or 2 week old 2

culture. Cell wall analyses yielded no conclusive results as to the cell wall type, thus YeAs was 3

considered Gram-indeterminate. No motility was observed when grown anaerobically in Orex-4 4

and Orex-4 (-cys) deeps (0.4% and 0.7% agar) supplemented with yeast extract and As(V). 5

Additionally, no flagella were seen in the microscopic examinations. Average cell length was 6 6

om with a range from 3 om to 10 om, while the average width was 100 nm and up to 200 nm 7

(Figures 3-5). Mineral deposition on the outer cell wall contributed to cell thickness. Electron 8

microscopy analysis revealed unusual rod shaped cell morphology and branching at the cell 9

terminus (Figure 3) when grown on Orex-4 with yeast extract and As(V). These branching 10

structures did not appear when the cells were grown with yeast extract only. Considerable 11

mineral deposition was observed on cell surfaces, and energy dispersive X-ray spectroscopy 12

(EDS) showed that the elemental composition of the mineral deposition was primarily arsenic 13

and sulfur (Figures 4 and 5), consistent with Raman spectroscopy data of mineral precipitates in 14

cultures. 15

Mineral precipitate characterization and production. Raman spectra collected from the 16

precipitate produced by YeAs in culture that had not been exposed to light exhibited a set of 17

prominent peaks in the 100 - 450 cm-1 region (Figure 6A). Consideration of the likely products 18

of arsenic reduction in the culture medium, combined with comparison of the experimentally 19

obtained spectra with previously published spectra suggested that the peaks were derived from 20

an arsenic chalogenide structure of the general form As4S4 (25). The lack of a principal peak at 21

220 cm-1 and a peak at 472 cm-1 indicated that the observed spectrum was not due to the presence 22

of either g or く polymorphs of sulfur (16). Absence of strong spectral features at 294 and 309 cm-23

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1 also precluded the presence of arsenic sulfides of the orpiment (As2S3) type (8). Tetra-arsenic 1

tetrasulfide exists in a number of well studied polymorphs, including realgar (g-As4S4), 2

pararealgar (light-induced polymorph of realgar), and a high temperature phase く-realgar (く-3

As4S4). Most instructive in structural discrimination are the A1, B1 and E modes of g- and く-4

realgar and the A' mode of pararealgar which gives rise to strong peaks between 340 and 365 cm-5

1 (25). These modes are dominated by ちAs-S vibrations, with smaller contributions from hAs-S-6

As and hS-As-S. The observed peak positions, 361 cm-1 (A1), 352 cm-1 (B1) and 342 cm-1 (E) all 7

indicated the presence of the く-realgar structure, the modes being shifted to lower wavenumbers 8

in g-realgar, and pararealgar having a single peak at 346 cm-1 in this region (Figure 6A). The 9

presence of a peak at 216 cm-1 was again more consistent with the B2 mode (principally hS-As-S 10

vibrations) of the く-realgar polymorph than of either realgar or pararealgar. The peak at 271 cm-1 11

could not be ascribed to く-realgar, but was consistent with an A' mode of pararealgar dominated 12

by hS-As-S vibrations, however there was no evidence for the stronger pararealgar doublet at 13

236/230 cm-1 which would have definitively indicated the presence of this polymorph. The 14

remaining peaks were consistent with く-realgar assignment although they are less instructive 15

when determining structure. Further confirmation of the く-realgar assignment could be gained 16

from following the photo-induced transformation of the putative structure to pararealgar. 17

Exposure to wavelengths shorter than 550 nm typically results in a rapid transformation of both g 18

and く polymorphs to pararealgar. When exposed to ultra-violet light, the YeAs precipitate proved 19

uncharacteristically photostable. Figure 6A also shows the FT-Raman spectrum of the く-realgar 20

precipitate following 12 hour UV-exposure. Ostensibly, there was little difference between the 21

dark and UV-light exposed spectra. However, a difference spectrum (Figure 6A) suggests that 22

limited transformation occurred during the exposure period. In this representation, peaks with 23

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negative intensity are reduced due to UV-light exposure, while positive peaks increase in 1

intensity. The four negative peaks were all consistent with く-realgar vibrations demonstrating 2

that some structure had been lost. Simultaneously, a number of positive peaks were observed 3

suggesting the formation of a new structure within the sample. The strongest peaks at 235 and 4

346 cm-1 were consistent with A' modes of pararealgar, being due to ちAs-As and ちAs-S 5

vibrations respectively (25). Other A' modes were evident at 275, 331, 201 and 146 cm-1. A" 6

modes were also evident at 190, 174, and 153 cm-1. Thus, there is evidence to suggest that 7

exposure of the precipitate to UV-light induces limited transformation to pararealgar. More 8

extensive transformation was observed upon exposure to sunlight for 2 weeks. The spectrum 9

collected from the precipitates after extended light exposure is shown in Figure 6B and 10

essentially represents a mixture of く-realgar and pararealgar. Strong contributions from く-realgar 11

were again evident at 361 cm-1 (A1 mode), 220 cm-1 (B2 mode) and 189 cm-1 (A1 mode). A 12

weaker B1 mode was also observed at 145 cm-1. The relative intensity of these peaks suggests 13

that a significant amount of く structure remains in the precipitate. However, a significant amount 14

of transformation occurred since strong peaks are observed for pararealgar at 345 cm-1 (A' 15

mode), a characteristic doublet at 234/232 cm-1 (A' mode), and a weaker contribution at 273 cm-1 16

(A' mode). 17

No く-realgar was produced by either T. celere or C. coolhaasii. In fact, very little to no 18

growth was seen with these organisms when grown with As(V), suggesting that the arsenic is 19

significantly more toxic to these strains. 20

Growth parameters and electron donor utilization. The doubling time for YeAs was 0.71 hours. 21

During late log phase there were 1.20x109 cells/ml for YeAs grown on Orex-4 with yeast extract 22

and As(V), 9.04x108 cells/ml when grown on Orex-4 (-cys) with yeast extract and As(V), and 23

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9.84x108 cells/ml on yeast extract only (Figure 7). These data suggest that YeAs is a 1

fermentative microorganism and does not rely on As(V) as a respiratory electron acceptor. 2

The temperature range at pH25˚C 6.8 for YeAs was 37˚to 75˚C with an optimal growth 3

temperature of 55ºC. YeAs grew at from pH55°C 6.0-8.0 and displayed optimal growth between 4

pH55°C 7.0-7.5. Growth was observed under anaerobic conditions only. 5

Based on Biolog plate results, YeAs was able to utilize the following substrates: fructose, 6

fucose, galactose, galacturonic acid, D-glucose-6-phosphate, palatinose, rhamnose, turanose, and 7

pyruvic acid. YeAs was not able to utilize the following substrates: acetate, formate, lactate, 8

succinate, malate, alanine, and valine. 9

G+C content and 16S rRNA gene analysis. The G+C content of YeAs was 35.0 mol% G+C. 10

Based on16S rRNA gene analysis, the sequence data revealed that YeAs branches within the 11

order Clostridiales. The phylogenetic tree indicates that the organisms most closely related to 12

YeAs include Thermobrachium celere (11), Caloramator coolhaasii (30), Caloramator 13

proteoclasticus (36), Caloramator viterbiensis (32), Caloramator indicus (9), Caloramator 14

fervidus (29), and Caloramator uzoniensis (Figure 8). YeAs had the highest sequence similarity 15

with C. proteoclasticus at 96.6%. 16

Enzyme detection for respiratory and detoxification arsenate reductases. Strain YeAs had no 17

detectable respiratory arsenate reductase, as judged by both spectrophotometric and in-gel assays 18

(Figure 9). YeAs demonstrated significant detoxification arsenate reductase activity with a 19

specific activity of 3.70 µmol NADPH/min/mg protein at 55ºC. This activity was glutathione-20

dependent, consistent with other reports of bacterial arsenic detoxification reductases (21). 21

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DISCUSSION 1

YeAs is an anaerobic, moderately thermophilic, arsenic-metabolizing bacterium that was 2

suspected to be novel based on its ability to reduce arsenic and biologically produce copious 3

amounts of the mineral く-realgar. Phylogenetic analysis revealed that YeAs is most closely 4

related to a group of organisms in the order Clostridiales, most specifically Thermobrachium 5

celere and six Caloramator sp. Table 1 illustrates general characteristics of 6 of the most closely 6

related organisms (detailed data for Caloramator uzoniensis are unpublished). 7

Although YeAs does not use As(V) as a terminal electron acceptor as indicated by 8

growth experiments, the reduction of As(V) by this organism is confirmed by two lines of 9

evidence: detection of arsenate reductase activity in cell extracts and the production of copious 10

amounts of く-realgar, a mineral composed of As(III) and sulfide (3). Our observation is the first 11

of bacterially mediated く-realgar production. Huber et al. (14, 15) reported a hyperthermophilic 12

archaeon capable of realgar production and characterized the mineral using X-ray diffraction, 13

SEM, and EDS. Additional reports have also mentioned realgar formation in the presence of 14

arsenate reducing bacteria (13, 27). Although realgar formation has been cited several times, in 15

no case was the polymorph of the realgar determined. く-realgar is normally produced abiotically 16

at high-temperatures above 250˚C (6). Additionally, arsenic sulfide minerals are typically 17

extremely photosensitive; however, the く-realgar produced by YeAs undergoes limited 18

transformation when exposed to UV-light. 19

One striking finding of these studies is that none of the phylogenetically-related 20

Thermobrachium and Caloramator isolates tested were able to metabolize arsenate and form く-21

realgar. In this case, phylogenetic relatedness makes a poor predictor of metabolic metal-22

transforming capability. Another unusual feature of this bacterium is the presence of structures at 23

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cell termini resembling attachment structures, or perhaps budding cells (2). This feature is not 1

commonly observed among the Bacteria and has not been reported for related genera of bacteria. 2

It is suspected that these structures are not flagella or pili as they appear branched along the 3

length of the structure, whereas flagella and pili tend to be single-stranded structures with an 4

origin along the cell surface and with no branching along the length. Because these interesting 5

structures only appear on cells grown with As(V) and are not seen on cells grown with yeast 6

extract only, it is also speculated that they could be inorganic mineral remnants. Further work is 7

proceeding to determine the origin, composition, and potential function of these structures. 8

The ability of YeAs to produce such a novel mineral has implications in several scientific 9

fields including those of nanotechnology (bionanomaterials), bioremediation, and exobiology. 10

YeAs could be considered as a solution for immobilizing arsenic in contaminated waters. The 11

rapid and efficient precipitation of arsenic by YeAs is a noteworthy mechanism that has the 12

potential for widespread use in these contaminated water systems. Additionally, く-realgar has the 13

potential to be utilized as an indicator of life in both characterized and uncharacterized extreme 14

environments. Because the abiotic formation of the high temperature phase mineral is 15

thermodynamically unfavorable (6), YeAs arsenic metabolism is unique and the mineral it forms 16

could be a significant ‘life signature’ in search for life on Mars and other worlds. 17

ACKNOWLEDGEMENTS 18

We thank the Alvord Ranch and the U.S. Bureau of Land Management for granting 19

permissions to the field site. This research was funded by a Research Infrastructure Improvement 20

grant (Award No. 0132626) from the National Science Foundation-Experimental Program to 21

Stimulate Competitive Research (J. Shreeve, PI, University of Idaho), and by NASA-EPSCoR. 22

R.N.L. was supported by a NASA-Idaho Space Grant Consortium Fellowship. Our work was 23

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facilitated by the Molecular Research Core facility (E. O’Leary-Jepson and M. Andrews) and the 1

Center for Biological Imaging (W. Siebold and C. Anderson). We thank Amanda Weaver for 2

critical review of the manuscript. 3

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8 9 10

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Characteristic YeAs T. celere C. coolhaasii C. proteoclasticus C viterbensis C. indicus C. fervidus Habitat

Hot spring

Several

locations

Thermophilic

granular sludge

Mesophilic granular

sludge

Hot spring

Thermophilic

aquifer

Hot spring

Geographical location

Alvord Basin, Oregon, USA

Several

locations

Wageningen, The

Netherlands

Montevideo,

Uruguay

Viterbo, Italy

Gujarat, India

New Zealand

Cell width (µm)

0.1-0.2 µm

0.5-0.8 µm

0.5-0.7 µm

0.4 µm

0.4-0.6 µm

0.6-0.8 µm

0.65-0.75 µm

Cell length (µm)

3-10 µm

1.5-13 µm

2-40 µm

2.4-4 µm

2-3 µm

10-100 µm

2-3 µm

Gram stain

Indeterminate

+

+

-

+

-

-

Spore formation

-

-

-

+

-

-

+

G+C content (mol%) Growth Temperature (°C)

35%

31%

31.7%

31%

32%

25%

39%

Optimal 55 66 50-55 55 58 60-65 68 Range 37-75 43-75 37-65 30-68 33-64 37-75 37-80 pH

Optimal 7.0-7.5 8.2 7.0-7.5 7.0-7.5 6.0-6.5 7.5-8.1 7.0-7.5 Range 6.0-8.0 5.4-9.5 6.0-8.5 5.5-8.7 5.0-7.8 6.2-9.2 5.5-9.0

Oxygen tolerance

Strict

anaerobe

Strict

anaerobe

Strict

anaerobe

Strict

anaerobe

Strict

anaerobe

Strict

anaerobe

Strict

anaerobe く-realgar production

+

-

-

ND

ND

ND

ND

Reference(s) This Study (11) (30) (36) (32) (9) (29)

Table 1. General characteristics of YeAs and the most closely related organisms. Strains include YeAs, Thermobrachium celere,

Caloramator coolhaasii, Caloramator proteoclasticus, Caloramator viterbensis, Caloramator indicus, and Caloramator fervidus

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Figure 1. Panel A: Map of a portion of the Mickey hydrothermal area. Murky Pot (M0290) is indicated and lies among several other mud vents and pools. Scale bar represents 10 m. Panel B: Murky Pot hot spring. Pool is turbid with flocculent materials. Scale bar represents 0.5 m.

B

A B

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Figure 2. Panel A: Culture of YeAs after 24 hours showing く-realgar production when grown with 0.5% yeast extract and 2.5 mM arsenic at 55°C. Panel B: Nomarski differential interference microscopy of YeAs showing long thin rods. Cells were in late log phase when the image was taken. Scale bar represents 2 µm.

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Figure 3. Transmission electron microscopy of YeAs cells grown under standard conditions with yeast extract and As(V). Panel A: Mineral (realgar) encrusted cell. Panel B: Unusual structures commonly found at cell termini.

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Figure 4. Panel A: Scanning electron microscopy of YeAs showing long thin cells and large く-realgar particle formed during growth. Panel B: Close-up of Panel A. Note evidence of layers running diagonally across the specimen.

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Figure 5. Energy-dispersive spectroscopy (EDS) analysis of mineral formed by YeAs at cell surface. Panel A: Tip of cell examined for elemental composition by EDS. Panel B: Results of EDS analysis showing significant arsenic and sulfide distribution on cell surface.

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Figure 6. Panel A: Raman spectra of mineral precipitates kept in the dark and those exposed to UV-light for 12 hours. Panel B: Spectrum of mineral precipitate following exposure to sunlight for two weeks, representing a mixture of く-realgar and pararealgar. In the accompanying table, peak positions for the polymorphs and for this study can be found.

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0.00E+00

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

1.20E+09

1.40E+09

0 5 10 15

Time (Hours)

Cells/m

lYeast extract and As(V)Yeast extract and As(V) (- cys)Yeast extract only

Figure 7. YeAs direct cell count data plotted against time. Note the similarity of growth between cells grown with and without As(V), suggesting that As(V) is not used as an electron acceptor.

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Figure 8. Phylogenetic dendrogram illustrating the phylogenetic position of YeAs. The scale bar indicates 0.10 changes per nucleotide. Tree was inferred by neighbor-joining using the Jukes and Cantor model to estimate evolutionary distances.

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Figure 9. Respiratory arsenate reductase activity stain showing the absence of activity in YeAs. 40 µg protein loaded per lane. Lanes: 1, Thermus; 2, YeAs.

1 2

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ledbetter et al. 2007 aem 1 biogenic mineral production by a novel

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