Wilmes et al.
SUPPLEMENTARY INFORMATION
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Supplementary Materials and Methods
For fluorescence in situ hybridization (FISH) and immunohistochemical detection
(IHCD), floating biofilms were cryo-embedded on site. Polystyrene Petri Dishes (60 mm
diameter; Becton Dickinson, Franklin Lakes, New Jersey) were prepared with
perforations running around the side walls approximately 3 mm from the base. On site,
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the bases of the dishes were covered with Tissue-Tek O.C.T. (Electron Microscopy
Sciences, Hatfield, Pennsylvania) pH 1 up to the perforations and placed in an ethanol
dry-ice bath until frozen. The dishes were then placed underneath the pellicles and raised
carefully from below to uniformly layer the biofilms onto the frozen Tissue-Tek. Excess
acid mine drainage (AMD) solution was allowed to drain off through the perforations and
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the holes were covered with Parafilm. One biofilm sample was taken per location per
date. The sampled biofilms were then overlaid with Tissue-Tek, frozen in the ethanoldry-ice bath and stored at –80 oC until further processing. Tailored blocks of embedded
biofilms (approximately 20 mm in length; Supplementary Figure 2) were sectioned (5, 20
or 30 m sections) on a Bright Model OFT cryomicrotome (Bright Instrument Co. Ltd.,
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Huntingdon, UK), layered onto glass microscope slides (ProbeOn Plus, Fisher Scientific,
Pittsburgh, Pennsylvania) and fixed in 2 % w/v paraformaldehyde (Electron Microscopy
Sciences, Hatfield, Pennsylvania) for 2 hrs at 4 oC. Rinsing and FISH were performed as
described previously (Bond et al., 1999). FISH was carried out with various broad-range
1
oligonucleotide probes [ARC915 (archaea; Amann et al., 1995), EUBMIX (bacteria;
Daims et al., 1999) and EUKMIX (eukarya; Baker et al., 2003)], and more specific
probes [LF655 (Leptospirillum groups II and III; Bond and Banfield, 2001), LF1252
(Leptospirillum group III; Bond and Banfield, 2001), TH1187 (Thermoplasmatales; Bond
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and Banfield, 2001)]. Probes were labeled with any of the following fluorochromes:
FITC, CY3 or CY5. For each sampling site, date and probe combination, six biofilm
coss-sections were prepared and hybridized. At C12, around 20 mm long transects were
prepared to cover the individual developmental stages (Supplementary Figure 2). For
simultaneous FISH and IHCD of Cyt579, 5 m biofilm thin-sections were permeabilized
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for 30 min in lysozyme solution (5 mg per ml of TE buffer) and for 15 min in proteinase
K solution (1 g per ml of TE buffer). Following FISH (using FITC and CY5 labeled
probes), a Cyt579-specific monoclonal antibody [raised in mice using the purified, native
Cyt579 protein (Singer et al., 2008)] was applied at appropriate titer for 1 hr followed by
washing each slide three times in 10 mM sodium phosphate, 150 mM NaCl solution at
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pH 7.4 (PBS) and once in purified water for 3 min each. Secondary antibody detection
was carried out in the dark for 1 hour with a Cy3-Goat Anti-Mouse IgG (H+L) conjugate
(Invitrogen, Carlsbad, California) at 1 mg per ml of 10 % v/v normal goat serum in PBS.
Biofilm thin-sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and
observed on a standard epifluorescent microscope (Leica DMRX, Leica Microsystems
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Inc., Bannockburn, Illinois) and/or on a confocal laser scanning microscope (CLSM;
Zeiss LSM 510 META, Carl Zeiss MicroImaging Inc., Thornwood, New York). For each
sampling site and date, six biofilm transects were prepared and hybridized. Seven zstacks were generated at random on the CLSM per microbial population, per
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development stage and per date. 3D image stacks were processed with IMARIS (Bitplane
AG, Zürich, Switzerland) and daime (Daims et al., 2006). The different populations were
quantified in 20-30 m thick z-stacks using the biovolume function in daime according to
the instructions in the User Manual. The exported biovolume data were analyzed using
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two-way analysis of variance (ANOVA) with replication using Microsoft Excel.
For transmission electron microscopy (TEM), mature biofilm samples from C12
were layered onto Isopore membrane filters (0.4 m HTTP type; Millipore Corporation,
Temecula, California). The filters were layered onto perforated Petri dishes, which
allowed for the precise sampling of biofilms and draining of excess AMD solution. Filter-
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supported biofilm samples were transferred into 150-200 m deep TYPE A aluminium
planchettes (Leica Microsystems GmbH, Wetzlar, Germany) that were sandwiched
against the flat side of TYPE B planchettes. The specimens were cryofixed in a Bal-Tec
HPM010 High Pressure Freezer (2100 bar, 5-7 msec; Bal-Tec Inc., Carlsbad, California;
Müller and Moor, 1984). Using the Leica automated freeze substitution system AFS
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(Leica Microsystems, Vienna, Austria), cryofixed specimens were freeze-substituted in
anhydrous acetone containing 1 % w/v osmium tetroxide/0.1 % w/v uranyl acetate and
infiltrated with Epon-Araldite following established protocols (McDonald, 2007).
Specimens were flat-embedded between two glass microscopy slides and polymerized at
60 °C for 1-2 days (Müller-Reichert et al., 2003). Resin-embedded biofilm samples were
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remounted under a dissecting microscope for precise orientation. 100-120 nm thin
sections were collected on Formvar-coated slot grids (Electron Microscopy Sciences) and
post-stained with 2 % w/v uranyl acetate in 70 % v/v methanol followed by either
Reynold’s or Sato’s lead citrate. Four serial sections of ~150 nm thickness of the biofilm
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in cross-section were imaged at a magnification of 3,000x on an FEI Tecnai 12 TEM
(FEI, Eindhoven, The Netherlands) using Kodak 4489 film. Films were scanned at 135
pixels per m. Individual images of each section were combined into a montage to cover
the entire biofilm cross-section. The four montages were superimposed using Adobe
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Photoshop and corresponded to a ~600 nm thick slab. Different cell types were
distinguished based on cell shape (bacterial spirilla versus archaeal cocci), size as well as
presence or absence of a pronounced S-layer (Bond et al., 2000). Bacterial, archaeal and
eukaryal cells were quantified and colored according to their taxonomic affiliation.
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Supplementary References
Amann RI, Ludwig W, Schleifer KH (1995). Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol. Mol. Biol. Rev. 59:
143-169.
Baker BJ, Hugenholtz P, Dawson SC, Banfield JF (2003). Extremely acidophilic protist
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from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening
sequences in their 16S rRNA genes. Appl. Environ. Microbiol. 69: 5512-5518.
Bond PL, Erhart R, Wagner M, Keller J, Blackall LL (1999). Identification of some of
the major groups of bacteria in efficient and nonefficient biological phosphorus removal
activated sludge systems. Appl. Environ. Microbiol. 65: 4077-4084.
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Bond PL, Druschel GK, Banfield JF (2000). Comparison of acid mine drainage microbial
communities in physically and geochemically distinct ecosystems. Appl. Environ.
Microbiol. 66: 4962-4971.
Bond PL, Banfield JF (2001). Design and performance of rRNA targeted oligonucleotide
4
probes for in situ detection and phylogenetic identification of microorganisms inhabiting
acid mine drainage environments. Microb. Ecol. 41: 149-161.
Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M (1999). The domain-specific
probe EUB338 is insufficient for the detection of all bacteria: development and
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evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22: 434-444.
Daims H, Lücker S, Wagner M (2006). daime, a novel image analysis program for
microbial ecology and biofilm research. Environ. Microbiol. 8: 200-213.
McDonald K (2007). Cryopreparation methods for electron microscopy of selected model
systems. Methods Cell Biol. 79: 23-56.
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Müller M, Moor H (1984). Cryofixation of thick specimens by high-pressure freezing. In:
Revel JP, Barnad T and Haggis GH (eds). The Science of Biological Specimen
Preparation for Microscopy and Microanalysis. Scanning Electron Microscopy Inc.:
O'Hara. pp 131–138.
Müller-Reichert T, Hohenberg H, O'Toole ET, McDonald K (2003). Cryoimmobilization
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and three-dimensional visualization of C. elegans ultrastructure. J. Microsc. 212: 71-80.
Singer SW, Chan CS, Zemla A, VerBerkmoes NC, Hwang M, Hettich RL et al (2008).
Characterization of cytochrome 579, an unusual cytochrome isolated from an iron
oxidizing microbial community. Appl. Environ. Microbiol. 74: 4454 - 4462.
5
Supplementary Table 1 pH and temperature measurement results of the AMD solutions
underlying the sampled biofilms at both locations on the three distinct sampling dates.
C12
C75
Date
pH
Temperature / oC
pH
Temperature / oC
6-Nov-06
1.01
39.5
1.03
40
2-May-07
1.14
39.3
1.17
44.4
7-Nov-07
0.96
24
0.91
41.5
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Supplementary Figure 1 Richmond Mine sampling sites: (I) C12, (II) C75. Direction of
acid mine drainage flow indicated by grey arrows.
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Supplementary Figure 2 Embedded biofilm. (a) Top view of cryo-embedded C12
biofilm in Petri Dish. Magnified area represents an excised transect block with the
individual developmental stages highlighted in color. (b) Cartoon of Petri Dish with
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individual developmental stages highlighted according to the color scheme used in a.
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Supplementary Figure 3 Leptospirillum group III in developmental stage 2 biofilm at
C12. (a) 3D CLSM FISH micrograph of Leptospirillum groups II and III. (b) FISH
micrograph of a typical Leptospirillum group III microcolony and (c) of single
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Leptospirillum group III cells. Micrograph color-coding: Leptospirillum group II,
Leptospirillum group III. Scale bars, 1 m.
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Supplementary Figure 4 Micrograph of immunohistochemical detection of Cyt579 in
C12 developmental stage 1 biofilm. This section was prepared identically to the C75
section in Figure 2c. Scale bar, 10 m.
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