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Supporting information for
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Extremophile microbiomes in acidic and hypersaline river sediments of
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Western Australia
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Shipeng Lu, Stefan Peiffer, Cassandre Sara Lazar, Carolyn Oldham, Thomas R. Neu, Valerian
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Ciobotă, Olga Näb, Adam Lillicrap, Petra Rösch, Jürgen Popp, and Kirsten Küsel
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Experimental procedures
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Sampling sites. The sampling sites were investigated during a field campaign to Dalyup
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Catchment at the end of the rainy season in September 2011. The surface water of West Dalyup
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River runs from north to south (Fig. 1A). The head of the West Dalyup River (site WH) is an
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excavated trench of one-excavator-bucket width and 2.1 m deep and exhibited low water flow.
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Green-colored algae-dominated biofilms were observed in the surface water. A 3-meter-deep
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piezometer was installed about 200 m north of the site WH and was determined to be upstream
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of the river. Surface water samples (sample WH-SW) and groundwater samples from the
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piezometer hole (sample WH-GW) were collected for further geochemical analyses. In the
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seepage zone of site WH, a gelatinous yellow colored muddy material (sample WH-YM) was
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found at the interface of surface water and trench side soil (Fig. 1B) approximately 5 meters
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downstream of the water source.
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Site DS was located about 3.6 km downstream from the headwater (Fig. 1A) The water
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depth at site DS was less than 20 cm. ‘Iron crust’-like sediment (Fig. 1C, sample DS-Crust) was
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observed on the river bed. The ‘iron crust’ was fragile and could be easily broken by pressing by
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hand. The ‘iron-crust’ was visually apparent from the sampling point to approximately one
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hundred meters downstream.
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For further chemical analyses all the water samples were filtered (0.45 μm), acidified if
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required (1vol-‰ 1M HNO3) and stored at low temperature (~ 4 °C). Water samples for isotopic
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analyses were filled in dark glass bottles (30 ml) without headspace and sealed with gas-tight
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screw caps. Fresh sediment samples were wrapped into two to three layers of zip-lock
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polyethylene bags and stored at ~ 4 °C for mineralogical analyses. Samples for molecular
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biological analyses were mixed with All-protect Tissue Reagent (Qiagen) in the field and stored
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frozen in the lab until processed.
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Geochemical characterization of the water samples. The pH and electrical conductivity
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of all the water and soil samples were measured on site with a pH/Cond 340i set (WTW). Metals,
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metalloids, cations and anions in water samples were analyzed using inductively coupled plasma
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optical emission spectrometry (ICP-OES) (Optima 3800XL; Perkin Elmer). Chloride anion and
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dissolved organic carbon (DOC) were measured by a commercial laboratory having experience
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with high salinity samples (ChemCentre, Australia). Fe(II) and total dissolved iron were
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determined in the acidic supernatant using the phenanthroline method (Tamura et al., 1974).
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Geochemical characterization, mineralogy and chemical composition analyses of the
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sediment samples. Soil pH and conductivity were determined in a slurry with a 1:5 ratio of soil :
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deionized water (Rayment and Higginson, 1992). Metals, metalloids, cations and anions were
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analyzed using either ICP-OES or ICP-MS. Dissolved sulfate concentration in sediments was
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measured using Barium-Chloride-Method (Tabatabai, 1974). Mineral phase examinations of
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samples WH-YM and DS-Crust were performed using Raman spectroscopy. The measurements
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were carried out with a commercial micro-Raman setup (HR LabRam inverse system, Jobin
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Yvon Horiba) using 532 nm radiation from a frequency doubled neodymium-yttrium-aluminum
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garnet laser. The laser beam of about 20 μW was focused on the samples by a Zeiss LD EC
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Epiplan-Neofluar 100X (numerical aperture, [NA] 0.75) microscope objective down to a spot
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diameter of approximately 0.7 μm. The spectral resolution was around 8 cm-1 and the acquisition
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time for each Raman spectrum varied between 30 and 300 s.
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The quantitative elemental composition was determined using x-ray fluorescence (XRF) in
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the Institute of Environmental Geology, Technical University of Braunschweig. Mineral phases
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were identified by powder X-ray diffraction using a D 5000 diffractometer (Siemens) equipped
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with a Co anode (Co Kα radiation). The diffraction patterns were measured in a range of 10-90°
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2θ and a step width of 0.02° 2θ (10sec/ step). The software DIFFRAC.SUITE (Bruker AXS
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GmbH) was used for the interpretation of diffractograms.
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The reactive and pedogenic iron in sample WH-YM were analyzed. For reactive iron
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measurement, 0.5 g of dried sample was transferred into 50-ml polyethylene vials containing 50
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ml 1M HCl (Wallmann et al., 1993; Kappler et al., 2004), followed by shaking at room
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temperature for 24 h. Fe(II) and total dissolved iron were determined in the acidic supernatant
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using the phenanthroline method. For extraction and determination of free pedogenic iron oxides,
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the reductive dissolution with Na-dithionite was used (Mehra and Jackson, 1960). The sample
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was dried and ground prior to analysis to reduce the particle size and increase the dissolution
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efficiency. Fe concentrations were analyzed using atom absorption spectrometry.
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Total DNA extraction and microbial community analyses. About 0.5 g wet material
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was used for each DNA extraction (Lueders et al., 2004) from sediment samples WH-YM and
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DS-Crust in triplicate. Retrieved DNA concentrations were determined by NanoDrop (Thermo
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Fisher Scientific, USA) and the DNA samples were sent for 16S rRNA gene pyrosequencing
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using 454 GS FLX+ platform (Research and Testing Laboratory, Texas, USA). The primer set
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28F and 519R was used to amplify the bacterial V1–V3 hypervariable regions, and primer set
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341F and 958R was used for archaeal V3-V5 regions (Suchodolski et al., 2009). The retrieved
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raw sequences were analyzed using Mothur (Schloss et al., 2009). Briefly, raw sequences were
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removed if they did not perfectly match the primer/barcode or were < 250 nt in length. Unique
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sequences from all samples were merged, chopped to achieve the same length. Then the
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sequences were aligned to the Silva reference alignment (Pruesse et al., 2007) as provided on the
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Mothur platform. The aligned sequences were preclustered and chimeras were removed using the
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Uchime algorithm implemented in Mothur. All remaining sequences were clustered into
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operational taxonomic units (OTU) at 0.03 and 0.10 distance cut-offs. The archaeal taxonomy
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was additionally checked and modified when necessary, by analysis of representative sequences
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for each OTU groups. This was carried out by aligning the representative OTU sequences using
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the SILVA web aligner (Pruesse et al., 2012) with reference 16S rRNA gene sequences of
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previously defined uncultured archaeal groups, and then constructing a phylogenetic tree in ARB
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(Ludwig et al., 2004). The percentage of coverage was calculated with the formula (1-[n/N] ×
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100, where n is the number of unique phylotypes and N is the total number of phylotypes. Both n
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and N values were obtained from Mothur.
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To obtain nearly-full length 16S rRNA gene sequences of the dominant phylogenetic
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groups for further analyses, an additional bacterial 16S rRNA gene clone library of sample DS-
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Crust was constructed using primers 27F and 1492R (Lane, 1991) as described previously (Lu et
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al., 2010). The phylogenetic analyses were conducted using Mega Ver4.0 and Neighbor-Joining
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method (Tamura et al., 2007).
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Quantitative PCR (qPCR) analyses. qPCR analyses of sediment samples WH-YM and
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DS-Crust were performed using 16S rRNA gene-specific primer sets for domains Bacteria and
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Archaea, genera Acidiphilium, Acidocella, Ferrovum and Acidimicrobium (Supporting
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information Table S1). The Acidocella-specific forward primer (Acido-LS-638, 5’-
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CTCTAGCTCACACGTATC-3’) was designed in this study and its specificity was verified using
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various online probe-match databases (Cole et al., 2009; Quast et al., 2013). Verifications were
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done by PCR with plasmids containing cloned PCR product, DNA from closely related strains,
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such as YE4-N1-5-CH (FN870350) from an acidic mine lake (Lu et al., 2010) and PFBC-3
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(KC590088) (Jones et al., 2013), and various environmental DNA extracts. An aliquot of 10-50
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ng of DNA extracts were used as templates for amplification with the Maxima SYBR Green
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qPCR Mastermix kit (Fermentas, Canada). Thermo-cyclings were performed with Mx3000P
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real-time PCR system (Stratagene, USA). Plasmids containing environmental clone sequences
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were used as standards. Acquisition of fluorescence data was performed at the end of each cycle.
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Dissociation curve analysis was performed to check the specificity of the qPCR products.
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Standard curves were linear from 5 × 102 to 5 × 108 gene copies per reaction. The amplification
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efficiency of Acidocella assay was 89% and the R2 value of the correlation curve was 1.000.
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Isolation of acidophilic bacteria. Six gram of the sample WH-YM was diluted with 2 ml
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0.7% (w/v) NaCl and homogenized by vortexing for 3 hours at ~1,000 rpm (Vortex-Genie 2
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shaker, Scientific Industries, United States). The sample was subsequently diluted ten-fold with
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0.7% (w/v) NaCl and 100 µl of the un-diluted and diluted samples were plated on each of the six
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kinds of selective solid overlay media used for autotrophic and heterotrophic acidophilic
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microorganisms (Johnson and Hallberg, 2007; Lu et al., 2010), including the yeast extract media
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with pH of 3 (YE3) and 4 (YE4) and other four high Fe(II) containing media (Lu et al., 2010).
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The plates were incubated at room temperature (~20 oC) in the dark and checked daily. Colonies
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appearing on the plates were observed and photographed using a stereo microscope (Olympus
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SZX10, Japan) and cell morphologies were examined under the brightfield-view of a microscope
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(Axioplan, Carl Zeiss, Germany). The picked colonies were transferred to freshly made media
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for at least five times and the cell morphology was monitored by microscopic examination. The
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obtained 16S rRNA gene sequences were searched against the online database BLAST (Johnson
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et al., 2008) to decide on their phylogenetic affiliations.
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Growth test of bacterial isolates in NaCl containing medium. One of the isolates
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(AusYE3-1) was tested for the aerobic growth in YE3 liquid medium (Johnson and Hallberg,
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2007). The YE3 liquid medium was a complex medium which contained 100 µM ferrous
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sulphate, 2% yeast extract and 10 mM fructose as carbon source and the final pH was adjusted to
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3. NaCl was added into the YE3 medium at concentrations of 1.5%, 3%, 4.5%, 6% and 7.5%
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NaCl (equal to 0.26, 0.51, 0.77, 1.03 and 1.28 M NaCl). The isolate cultures were incubated at
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15 °C or 30 °C, and the optical density was measured at 600 nm wavelength. Triplicates of each
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treatment were prepared and the growth tests were repeated three times. In addition, the isolate
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was tested for its anaerobic growth and ferric reduction capacity in YE3 medium with or without
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3% NaCl, 10 mM fructose and 35 mM ferric sulphate. The medium was flushed with nitrogen
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gas in serum bottles (Wheaton, United States) for more than 20 min and closed by rubber
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stoppers and aluminium caps. Active cultures were then added into the medium using needles.
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Actively grown cultures were stained with nucleic acid stain CYTO13 (Molecular Probes),
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followed by observation and microphotograph using an epifluorescence microscope (Axioplan,
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Carl Zeiss, Germany) equipped with a mercury arc lamp.
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Three Acidiphilium strains, strain JF-5 (Y18446) (Küsel et al., 1999), strain SJH
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(AY040740) (Hallberg and Johnson, 2001) and YE3-D1-35 (FN870339) (Lu et al., 2010), were
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tested for their heterotrophic growth in the salt-containing YE3 medium and their anaerobic
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growth in anoxic 3% NaCl YE3 medium.
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Confocal laser scanning microscopy (CLSM). For a better visualization of the cell chains
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structure, the bacterial isolate AusYE3-1 grown at 3% NaCl was stained with SYTO 9 nucleic
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acid stain and FM 4-64 membrane stain (Molecular Probes). Samples were examined by CLSM
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(TCS SP5X; Leica) using a white laser source, an upright microscope, and a 63x 1.2 NA water
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immersion lens. Images were recorded as a single scan and the parameters were as follows:
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SYTO9, excitation at 500 nm, emission at 515-570 nm; FM4-64, excitation at 500 nm, emission
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at 680-780 nm. The data set was subjected to blind deconvolution using Hygens ver. 14.10
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(Scientific Volume Imaging). Images were projected in Imaris ver. 8.0.1 (Bitplane) and printed
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from Photoshop CS6 (Adobe).
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Nucleotide sequence accession numbers. The 16S rRNA gene sequences of the clone
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library of sample DS-Crust have been deposited in the EMBL database under accession number
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of HG975547 to HG975585. Bacterial and archaeal 16S rRNA gene pyrosequencing reads have
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been deposited in the EMBL database under study accession number of PRJEB7755.
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Table S1 Primers used for qPCR detecting the two abundant bacterial groups.
Target
Bacteria
Acidiphilium
Acidocella
Ferrovum
Acidimicrobi
um
Archaea
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a
Primer
Sequence (5’-3’)
Uni-338F-RC
ACT CCT ACG GGA GGC AGC
Uni-907R
CCG TCA ATT CMT TTG AGT TT
ACD840
CGA CAC TGA AGT GCT AAG C
Uni-338F-RC
ACT CCT ACG GGA GGC AGC
Acido-LS-638
CTC TAG CTC ACA CGT ATC
Uni-338F-RC
ACT CCT ACG GGA GGC AGC
Ferrovum643F
ACA GAC TCT AGC TTG CCA GT
Uni-338F-RC
ACT CCT ACG GGA GGC AGC
Amf995-C25
CTC TAC GGC TTT TCC CAA CAT G
Uni-907R-RC
AAA CTC AAA KGA ATT GAC GG
Arch806F
ATT AGA TAC CCS BGT AGT CC
Arch958R
YCC GGC GTT GAM TCC AAT T
Amplicon
length (bp)
AFC: Amplification fluorescence collection.
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8
Annealing
tem. (oC)
AFCa
tem. (oC)
571
57
78
505
61
78
293
57
78
323
61
78
110
53
79
196
56
80
Reference
Lane, 1991
Lane, 1991
Bond and
Banfield, 2001
Lane, 1991
This study
Lane, 1991
Heinzel et al.,
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Lane, 1991
Cleaver et al.,
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Lane, 1991
Takai and
Horikoshi, 2000
Delong, 1992
Standard
clone (acc. no.)
HE604015
HE604018
HG975566
HE604015
HE604007
Clone library
based plasmid
mixture
156
Table S2 Growth tests of different Acidiphilium strains in NaCl-containing media under
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oxic and anoxic cultivation conditions at 30 °C.
Acidiphilium strains
(Accession No.)
AusYE3-1
JF-5 (Y18446)
SJH (AY040740)
D1-35 (FN870339)
0
+
+
+
+
NaCl concentration (% w/v)
Oxic
Anoxic
1.5 3.0 4.5 6.0 7.5
0
3.0
+
+
+
+
+
- NDb
+
+
+
+
a
ND
+
+ (+)
+
ND
+
+
+
-
158
159
a
Slow cell growth observed.
160
b
ND: not determined.
161
162
9
163
Fig. S1 Selected Raman spectra of samples WH-YM (A) and DS-Crust (B). Jarosite and
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goethite were observed in sample WH-YM, while schwertmannite and jarosite were main
165
minerals in DS-Crust. Only spectra of iron minerals are presented and spectra of graphite,
166
anatase, rutile and quartz are not showed. R stands for representative references and S for
167
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168
169
10
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Fig. S2 Quantitative PCR analyses of DNA extracts from the ‘yellow mud’ sample WH-
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YM and ‘iron crust’ sample DS-Crust. Total bacterial 16S rRNA genes and the
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Alphaproteobacteria Acidiphilium and Acidocella groups were detected. Archaeal 16S
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174
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of 5 × 102 gene copies per reaction.
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Fig. S3 Phylogenetic tree of Acidiphilium- and Acidocella-related sequences showing the
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close relationship of 16S rRNA gene sequences obtained from ‘iron crust’ sample DS-
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Crust and a representative strain (AusYE3-1) isolated from ‘yellow mud’ of sample WH-
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YM. The tree was constructed using neighbor joining method. The nearly-full 16S rRNA
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gene sequences from the clone library were used for the tree construction instead of the
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short reads from 454-pyrosequencing. GenBank sequences accession numbers are shown
183
in parentheses and sequences from this study are shown in bold. The Archaea
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Methanosarcina barkeri (AJ012094) was used as an out-group (not shown). Scale bar
185
shows 0.1 change per nucleotide position.
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186
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13
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Fig. S4. Ferric iron reduction test of Acidiphilium strain AusYE3-1 in acidic YE3
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medium. (A) Beginning of the incubation. (B) AusYE3-1 was able to reduce Fe(III)
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medium (left tube) after about 2 weeks of incubation. (C) The Fe(III) reduction was not
192
observed after more than 2 months incubation in the present of 3% NaCl.
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