Supporting Information - PNAS · 2009. 10. 7. · mirrors, with a graded multilayer coating the first one, thus func-tioning both as monochromator and vertical focusing optics. This

Supporting InformationReith et al. 10.1073/pnas.0904583106SI Materials and MethodsAu(III) Accumulation Experiments. C. metallidurans CH34 was ob-tained from the German Culture Collection (DSMZ-No. 2839).Cells were grown in PME medium containing: Peptone, 5 g/L; meatextract, 3 g/L; pH after autoclaving 6.6; medium contained: K (210mg/L); concentrations of all other elements were below the limitsthe detection of the inductively coupled optical emission spectrom-eter (ICP-OES; Spectro Arcos): Ca (10 mg/L), Na (100 mg/L), S (10mg/L), Al (5 mg/L), B (5 mg/L), Fe (10 mg/L), P (10 mg/L), Si (5mg/L), and the inductively couple plasma mass spectrometer [ICP-MS, Agilent 7500ce ICP-MS; Agilent Technologies]: Cu (5 �g/L),Zn (6 �g/L), Ga (1 �g/L), Ge (1 �g/L), As (0.8 �g/L), Se (5 �g/L),Sr (2 �g/L), Y (2 �g/L), Nb (2 �g/L), Mo (5 �g/L), Ru (0.4 �g/L),Pd (5 �g/L), Ag (1 �g/L), Cd (0.5 �g/L), Sn (0.4 �g/L), Sb (0.5�g/L), Cs (2 �g/L), Ba (4 �g/L), La (0.1 �g/L), Ce (0.1 �g/L), Pr(0.1 �g/L), Nd (0.1 �g/L), Sm (0.1 �g/L), Eu (0.1 �g/L), Gd (0.1�g/L), Tb (0.1 �g/L), Dy (0.1 �g/L), Ho (0.1 �g/L), Er (0.1 �g/L),Tm (0.1 �g/L), Yb (6 �g/L), Lu (0.1 �g/L), Hf (1 �g/L), Ta (5 �g/L),W (0.5 �g/L), Re (0.1 �g/L), Pt (0.2 �g/L), Au (0.4 �g/L), Tl (0.4�g/L), Pb (0.6 �g/L), Bi (0.2 �g/L), Th (0.5 �g/L), U (0.1 �g/L).Au(III) accumulation experiments were conducted in triplicate foreach condition, i.e., cell status (viable, inactive, or dead plus abioticcontrol), pH (pH 5.0, 6.0, 7.0, and 8.0; pH adjusted with analyticalgrade 5 M HCl or NaOH solution), and time (6 and 144 h) byinoculating 10-mL centrifuge tubes containing 2.5 mL growthmedium with 5.5 � 104 cells/mL. Cultures were incubated for 16 hat 30 °C on a shaking incubator at 100 rpm (revolutions/min). Cellswere harvested by centrifugation at 4,500 � g for 15 min, and thesupernatant was decanted. To produce metabolically inactive cells,the cultures were treated with 1 mL 2.5% (wt/vol) formalin for 2 hat room temperature (1), after which they were washed twice insterile 0.9 wt% NaCl solution, and centrifuged at 4,500 � g for 10min. The effectiveness of the formalin treatment was determinedusing O2-respiration measurements on treated cells amended with5 mL fresh medium for up to 24 h, using a dissolved oxygenelectrode (YSI) calibrated with air-saturated water (i.e., 100%saturation). While O2 saturation levels measured in this mannerwere below the detection limit of 3.3% after 1 h of incubation foruntreated samples, the O2 saturation in samples treated withformalin remained above 80% for the 24 h measuring period. Deadcells were generated by autoclaving for 1 h at 121 °C.

Five milliliters of pH-adjusted (pH 5.0, 6.0, 7.0, and 8.0) PME-medium were added to each of the tubes, and Au(III) was addedto the chosen final concentration of 50 �M from a liquid 50 mMstock solution prepared by dissolving HAuCl4 � 3H2O in deionizedwater. The data shows that the Au concentration was more than 3orders of magnitude higher relative to the concentrations of othermetals in the medium. Hence competition for cell wall binding sitesduring the initial sorption stage is negligible, and interference of Ausorption by other metals can be ruled out. Thermodynamic dataindicate that aqueous Au(III) in the solutions existed as a negativelycharged square planar complex, with [AuCl4]� predominant underacidic conditions, [Au(OH)4]� predominant under basic condi-tions, and mixed hydroxychloride complexes [AuClx(OH)4-x]� dom-inating under near-neutral conditions (2). Tubes were incubated ona shaking incubator for 6 and 144 h at 100 rpm and 30 °C in the dark.Cells were harvested by centrifugation at 4,500 � g for 15 min. Aftercentrifugation, the supernatant was decanted, filtered through a0.2-�m microfilter, and acidified with analytical grade HCl. Auconcentrations were measured in the filtrate using an Agilent7500ce ICP-MS (quantification limit 0.4 ppb; Agilent Technologies)connected to a CETAC ASX-500 autosampler with He as collision

gas. pH was measured at the start, after 6 h, and after 144 h usinga pH meter and probe (Activon). This data shows that in inactive,dead, and abiotic control experiments, no significant changes to thepH occurred, thus indicating a pH dependency of Au uptake. Inactively metabolizing cell incubations, the pH was 5.8, 6.5, 7.0, and7.7 in experiments with starting pH of 4.0, 5.0, 6.0, and 7.0,respectively. After 144 h, the pH approximated 7.0 independent ofthe starting pH.

In contrast to metabolically inactive and dead cell experiments,cell density and biomass increased in experiments with metaboli-cally active cells. To account for this, the Au adsorption results werenormalized using the cell mass present at the end of the incubation.Viable cells were counted using the drop plate method (3) onPME-agar plates that contained 5 g/L peptone, 3 g/L meat extract,and 15 g/L agar.

Metabolically active C. metallidurans cells for synchrotron X-rayfluorescence (SXRF) experiments were grown and harvested asdescribed above at pH 7.0. Cells were harvested after 1, 10, and 30min and 1, 6, 48, 72, and 144 h incubation in medium spiked withAu(III). For bulk XANES measurements, cells were filteredthrough a 0.45-�m filter, which was air-dried in a dust-free envi-ronment, and stored at �20 °C until analyses at the StanfordSynchrotron Radiation Laboratory (SSRL). For �XRF and�XANES measurements at the Advanced Photon Source (APS)and European Synchrotron Research Facility (ESRF), the cellpellets were resuspended in 0.1 mL 0.9 wt% NaCl solution anddiluted to a factor of 1:1,000 with sterile double-deionized water 30min before analysis. Dilution (2 �L) was transferred to siliconnitride membranes (Silson Ltd) and air-dried in a dust-free envi-ronment.

�XRF-Mapping, Bulk, and �XANES Data Collection and LCF. The APSis a 7-GeV synchrotron that operates in top-up mode resulting in aconstant current of 100 mA. The 2-ID-D has an Si(111) mono-chromator, and X-rays were focused using a Fresnel zone plate witha focal length of 0.1077 m at 11.93 keV, resulting in a focused beamspot of 250 � 190 nm. An order-sorting aperture intercepted higherdiffraction orders and, in conjunction with a central stop, blockedthe unfocussed beam. SXRF data were collected at an incidentbeam energy of 11.93 keV. A single element Ge energy-dispersivedetector was located at �90 ° to minimize detection of the scatteredbeam.

The ESRF is a 6.03 GeV ring with a maximum current of 200 mAwhen operating in uniform bunch mode. ID22NI is an undulatorbeamline with X-ray optics designed to focus the beam to nano-meter size in pink beam mode operation (4). The beamline has aSi mirror at an incident angle about 2.6 mrad for high harmonicrejection. In the Kirkpatrick-Baez geometry used in this experi-ment, the focusing system comprises of two elliptically shapedmirrors, with a graded multilayer coating the first one, thus func-tioning both as monochromator and vertical focusing optics. Thisresults in a photon flux of up to 1012 photon/s at energies between15 and 17 keV. The beam was focused to 120 � 150 nm, with anincident energy of 17.5 keV. The sample was mounted on aXY-piezo-nano-positioner stage and scanned at the focal plane,while the emitted X-ray fluorescence data were measured with asingle-element Si Drift detector.

At both 2-ID-D and 22IDNI, full fluorescence spectra weremeasured at each pixel, compared to just collecting regions-of-interest, for both SXRF and �XANES data. SXRF data werequantified using PyMCA (22IDNI, ESRF) (5) and GeoPIXE II(2-ID-D, APS) (6). For the ESFR data, beamline parameters were

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calibrated based on the NIST 1577b bovine liver standard, and thedata were normalized such that Si (from the Si3N4 window used asa sample support) had a concentration of 4 wt% (2500 ng cm�2).The 2-ID-D SXRF data were calibrated using the NIST 1832standard. GeoPIXE II utilizes the Dynamic Analysis method toproject quantitative elemental images from SXRF data, and quan-titative maps from both data sets were constructed using GeoPIXEII (7).

The speciation of accumulated Au was assessed using XANESspectroscopy and LCF. Bulk XANES measurements on C. metal-lidurans cell pellets were conducted at the SSRL with the SPEARstorage ring containing between 80 and 100 mA at 3.0 GeV. GoldL3-edge data were collected on the structural molecular biologyX-ray absorption spectroscopy (XAS) beamline 7–3 operating witha wiggler field of 2 T. A Si(220) double-crystal monochromator wasused. Beamline 7–3 is equipped with a rhodium-coated verticalcollimating mirror upstream of the monochromator. Incident andtransmitted X-ray intensities were monitored using Ar- or N2-filledionization chambers. X-ray absorption was measured at the AuL3-edge in fluorescence mode using a 30-element Ge array detec-tor. During data collection, samples were maintained at a temper-ature of �10 K using an Oxford Instruments liquid He flowcryostat. For each sample, the energy was calibrated by referenceto the absorption of Au foil measured simultaneously with eachscan, assuming a lowest energy inflection point of 11.919 keV. XASdata reduction was performed with the EXAFSPAK suite ofcomputer programs (http://ssrl.slac.stanford.edu/exafspak.html),employing a Gaussian pre-edge function, and a weighted polyno-mial spline with normalization correction to extract the EXAFSoscillations.

The 2-ID-D beamline set-up also allowed for the collection of�XANES data of individual bacterial cells. Instead of collectingregion of interest, full SXRF spectra were collected at each energypoint from 11.88 to 11.98 keV, with a step of 0.5 eV. The intensityof the fluorescence line of interest, in this case the Au L� line, wasextracted from profile fits in GeoPIXE II. These �XANES spectrarequired smoothing (10 iterations of interpolative smoothing). Thedata were normalized by subtracting the pre- and post-edge back-ground of the metal foil collected at 2-ID-D, such that this spectrummatched that of the Au foil EXAFS spectrum collected at theSSRL. These normalization parameters were then applied to allspectra collected at 2-ID-D, and the normalization was verifiedusing MBACK (8).

Peak- and LCF of bulk- and �XANES data were conductedusing the Athena software (9, 10). Individual scans from thedifferent experiments were calibrated and aligned using the Au foilspectra as reference. C. metallidurans and Desulfovibrio sp. spectrawere fitted with linear combinations of standard spectra. Standardspectra were obtained from a number of at least 99.9% pure modelcompounds measured in solid form in transmission mode at boththe SSRL 7–3 and APS 2-ID-D beamlines, i.e., Au(III)-chloride(HAuCl4.3H2O), Au(III)-thiocyanate [KAu(SCN)4], Au(I)-chloride (AuCl), Au(I)-cyanide (AuCN), Au(I)-thiosulfate[Na3Au(S2O3)2.2H2O], Au(I)-sulfide (Au2S), Au(I)-thiomalate,and Au(0)-foil (Alfa Aesar or Sigma-Aldrich). These model com-pounds were chosen as reference because they represent the moststable oxidation states of Au, i.e., Au(0), Au(I), and Au(III), andwere used to qualitatively illustrate the environment expected to befound in the samples (2). The model compounds, except for Au foil,were ground and mixed with boron nitride, and then packed into1-mm path length plastic holders with Kapton tape windows beforethe measurement to achieve an edge step near unity in transmission.

SEM and TEM of Au Grains and C. metallidurans Cells. Ten Au grainswere obtained under field sterile conditions from the Prophet GoldMine in Queensland, Australia. Each gold grain was repeatedlywashed (10 times) onsite with and stored in sterile 0.9 wt% NaClsolution. Samples were transported on ice to the laboratory. Au

grains were again washed in sterile double-deionized water toremove salt and air-dried in a dust-free environment. Grains weremounted on adhesive carbon tape attached to sample holders andstudied uncoated using a focused ion beam electron microscope(Helios NanoLab DualBeam; FEI) equipped with a light elementEDXA-detector at Adelaide Microscopy.

Ultra-thin sections of C. metallidurans cells from the experimentswere imaged and mapped using a Phillips CM-200 TEM equippedwith a light element EDXA-detector operating at 200 kV. C.metallidurans cells were fixed for 16 h (SEM/TEM fixative), post-fixed with 2% (vol/vol) osmic acid for 1 h, dehydrated with a seriesof 25, 50, 75, and 100% ethanol solutions, infiltrated with 1:1mixture of 100% ethanol and epoxy resin for 1 h, followed by two1-h 100% resin infiltration steps (Procure Resin; ProSciTech).Samples were then imbedded in fresh resin, which was allowed topolymerized at 70 °C for 24 h. Sections were cut using an UltracutE microtome (Leica) to a thickness of 70 nm using a diamond knifeand collected on Formvar C-coated 200-mesh Cu grids.

Transcriptome Microarray Analysis of Responses to Au(III)-Complexesin C. metallidurans. The MIC was determined in triplicate as thelowest concentration inhibiting bacterial growth on solid TrisMMcontaining 2 g sodium gluconate/L and 20 g agar/L (11). Apreculture was incubated at 30 °C, 250 rpm, for 48 h, then diluted1:20 in fresh medium and incubated for 24 h at 30 °C and 250 rpm.This 24-h culture was used for streaking onto plates containing0–2.5 �M Au(III). The plates were incubated at 30 °C for 5 days,and cell growth was monitored. Further in vivo experiments inliquid TrisMM and PME-media showed that cells were able to growin the presence of up to 50 �M of Au(III)-complexes, and viablecells could be detected after incubation in media containing up to100 �M Au(III). Based on these experiments, four Au(III) sets ofconditions were chosen for transcriptome microarrays, i.e., 10, 50,and 100 �M for 10 min, and 50 �M Au(III) for 30 min.

Cultivation of C. metallidurans CH34 subcultures for RNAextraction after Au(III)-chloride challenge was conducted in trip-licate in Klett flasks containing 45 mL TrisMM (plus 2 g sodiumgluconate/L). Flasks were inoculated with 1% of preculture (450�L) and then incubated at 30 °C on a rotary shaker (250 rpm).When OD600 � 0.58 was reached (corresponding to a cell-densityof 4.5 � 108 cfu/mL), each culture was divided into two 20-mLflasks. One flask was used as condition [i.e., challenged with theappropriate amount from a 50 mM Au(III) stock solution] while theother flask served as control [i.e., water added instead of Au(III)stock solution]. The flasks were incubated for 10 or 30 min,harvested by centrifugation for 1 min at 10,000� g, the supernatantwas decanted, cell pellets were flash-frozen in liquid N2, and storedat �80 °C until RNA extraction.

RNA-stabilization was conducted by adding 300 �L RNALaterII solution (Ambion or Applied Biosystems) to each of the frozencell pellets, which were then incubated at room temperature for 1 h.Bacterial cells containing stabilized RNA were centrifuged for 2min at 10,000 � g, and the supernatant was discarded. Lysis wasperformed by suspending the cell pellets for 10 min in a 3 g/Llysozyme (Sigma-Aldrich) solution at room temperature. TotalRNA extraction was conducted using the SV Total RNA Isolationsystem (cat# Z3100; Promega). RNA quantity was assessed usingwith a NanoDrop 1000 spectrophotometer (Thermo Scientific),and RNA quality was measured as RNA integrity number (RIN),with a 2100 Electrophoresis Bioanalyzer (Agilent Technologies).Only RNA with a RIN �8.5 was used for microarray analysis (12,13).

The draft genome sequence (November 2003) of C. metalliduransCH34 was used to design 60-mer aminosilane-modified oligonu-cleotide probes corresponding to the predicted 6.205 ORFs. Theseoligonucleotide probes were synthesized by Eurogentec S.A. andspotted in triplicate onto glass slides (UltraGPS; Corning) using aMicroGrid system (BioRobotics). The spotted slides were cross-



linked and placed in the presoaking solutions from the Pronto Kit(Promega).

Ten micrograms RNA were reverse-transcribed using the Prontokit (Promega) following the manufacturer’s instructions. Two-colorlabeling, green for control samples and red for condition [i.e.,Au(III) present] samples, was performed using respectively, Cy3-dCTP and Cy5-dCTP nucleotides (Amersham BioSciences). La-beled cDNA was resuspended in the universal hybridization buffer(Pronto kit; Promega), mixed, and added to the spotted slide forovernight hybridization at 42 °C in a HS4800 Pro hybridizationstation (Tecan). Afterward, the slide was washed according toPronto kit’s protocol (Promega). Slides were scanned (at 532 and635 nm) using the GenePix Personal 4100A microarray scanner(Molecular Devices).

Microarray spot-signals were analyzed using the GenePix Prov.6.0.1 software (Molecular Devices) and flagged according tobuilt-in quality criteria. Quality check was performed for each arrayusing the ArrayQuality package from BioConductor (14). Rawmedian intensity data were imported into R version 2.7.0 forstatistical analysis using the LIMMA package version 2.15.15 asavailable from BioConductor (15). Raw data were background-corrected based on convolution of normal and exponential distri-butions with an offset of 50 (16). Data were normalized within eacharray using the printing-tip loess normalization algorithm (17). Thein-slide replicate correlations were calculated using the duplicatecorrelation function in the LIMMA package (18). The log expres-sion values were fitted to a linear model and moderated t-statisticswere calculated using empirical Bayes method (19). P values werecorrected for multiple testing using the Benjamin and Hochberg’smethod to control the false discovery rate (20). Analyses of clustersof orthologous groups of proteins (COGs) were conducted usingthe procedures outlined in Tatusov et al. (21). Circular plots were

constructed using the Circos library v. 0.37. The full description ofthe array analysis platform and the complete array data have beendeposited at the Gene Expression Omnibus web site (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE14049.

Reverse Transcription PCR (RT-PCR) of Au-Specific Genes (Rmet�4682to Rmet�4687). C. metallidurans CH34 and �rpoQ deletion mutantcells (DN482) (22) were cultivated as described above for transcip-tome analyses. Cells were challenged with 50 �M Au(III) for 10 minat pH 7.0. Control incubations with double-deionized H2O for 10min under otherwise identical conditions were used. Total RNAwas isolated using RNeasy Mini kit (Qiagen) according to themanufacturer’s instructions. Extracted RNA was treated twice withDNase. RNA concentration was determined photometrically, andRNA quality was checked on formamide gels (23). To excludeexperimental artifacts resulting from DNA contamination, onlyRNA was used that did not generate product in PCRs withchromosomal primers without a previous RT reaction. For RTreaction, 1 �g total RNA and 0.1 �g hexamer primers wereincubated at 65 °C for 5 min and snap-cooled on ice. After additionof 0.5 mM each of dATP, dGTP, dTTP, and dCTP, 10 mM DTT,and 100 U of reverse transcriptase (SuperScript II) in reactionbuffer (Invitrogen) RT proceeded for 10 min at room temperature,followed by 1 h at 50 °C. After finishing the RT reaction, theenzyme was inactivated at 70 °C for 10 min. The resulting cDNAwas amplified by PCR (50 �L) with 1 �L template cDNA, 0.2 pmoleach primer (Metabion), 0.2 mM dNTP mix (GE Healthcare), 1 UTaq polymerase (Roche Diagnostics); further details for PCRprotocols and primer sequences are available on request. As anendogenous control, rpoZ was used. All cDNA displayed the sameexpression level when amplified with primers for the gene rpoZ. Ano-template control was performed under identical conditions asfor the target genes. All experiments were conducted in replicate.

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Fig. S1. (A) Viable cell numbers of C. metallidurans at the start, after 6 and 144 h of exposure to Au(III) of untreated (biologically active, darker color) andformalin-treated (biologically inactive, lighter corresponding color) C. metallidurans cells incubated in PME medium at pH 5.0 to pH 8.0. (B) Viable cell numbers of C.metallidurans CH34 incubated for up to 180 min in TrisMM amended with 0 (�), 10 (Œ), 50 (�), and 100 () �M Au(III) added at a cell density of OD600 � 0.58. Cells wereincubated on a shaking incubator at 30 °C; error bars represent the standard deviation of the triplicates and lie within the area of the symbol, if not visible in the graph.



Fig. S2. Accumulation of Au(III) by C. metallidurans, quantitative �XRF-maps for Au, Ca, Cu, Fe, S, and Zn distribution, and overlay false color quantitative �XRF-mapof the distribution of Au (red), Zn (blue), and Cu (green) in and around cells after 10-min (A) and 30-min (B) exposure to 50 �M Au(III). Quantified regions are markedin the image, and concentrations, calculated errors, and concentration ranges are given.



Fig. S3. Accumulation of Au(III) by C. metallidurans, quantitative �XRF-maps for Au, Ca, Cu, Fe, S, and Zn distribution, and overlay false color quantitative �XRF-mapof the distribution of Au (red), Zn (blue), and Cu (green) in and around cells after 6 h exposure to 50 �M Au(III). Quantified regions are marked in the image, andconcentrations, calculated errors, and concentration ranges are given.



Fig. S4. Accumulation of Au(III) by C. metallidurans, quantitative �XRF-maps for Au, Ca, Cu, Fe, S, and Zn distribution in and around cells after 72 h exposure to 50�M Au(III). Quantified regions are marked in the image, and concentrations, calculated errors, and concentration ranges are given.



Fig. S5. Accumulation of Au(III) by C. metallidurans, quantitative �XRF-maps for Au, Ca, Cu, Fe, S, and Zn distribution in and around cells after 144 h exposure to 50�M Au(III). Quantified regions are marked in the image, and concentrations, calculated errors, and concentration ranges are given.



Fig. S5 (continued).



Fig. S6. (A) Transmission electron micrograph of C. metallidurans CH34 cell section after exposure to 100 �M Au(III) for 144 h; (B–D) X-ray maps of Au, S, and Ca ofsectioned C. metallidurans obtained using EDX analyses.



Fig. S7. Expression analyses of chromosome 1 (A), chromosome 2 (B), plasmid pMOL28 (C), and plasmid pMOL30 (D) from C. metallidurans CH34 after induction withAu(III). The innermost circle is GC skew plot in which the (G-C)/(G�C) ratios are shown in sliding windows of 500 nucleotides with a window overlap of 250 nucleotides.The next circle is a GC deviation plot and represents the mean centered GC content (purple, above mean; orange, below mean) using a window size of 500 nucleotidesand a 250-nucleotide window overlap. The next circle (numbered 1) correspond to metal density of upregulated genes with the metals tested in a previous study (i.e.,Ni2�, Zn2�,Cu2�,Cd2�, Pb2�,Hg2�, andCo2�) (22).Thenext fourcircles (numbered2–5)correspondtotheresultsgeneexpressionfor thefour inductionconditions listed.Significant (�2-fold) downregulation is shown in green; upregulation is shown in increasing shades of red corresponding to the degree of increased expression. Thecircles (numbered 6) on the outside display the genes in functional categories according to the COG color scheme (from NCBI, Bethesda, MD; http://www.ncbi.nlm.nih.gov/COG/grace/fiew.cgi), with genes in the outer circle oriented clockwise, and genes in the inner circle oriented anti-clockwise. Metal resistancegene clusters are shown as black bars.



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Supporting Information - PNAS · 2009. 10. 7. · mirrors, with a graded multilayer coating the first one, thus func-tioning both as monochromator and vertical focusing optics. This