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SUPPLEMENTARY INFORMATION
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DNA extraction from Freshwater quarry mats
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DNA was extracted from mat material using the Mo-Bio Power Soil DNA
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extraction kit, following the protocol for difficult-to-lyse cells (heating tubes to 70°C for
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10 minutes after addition of solution C1). DNA was quantified by electrophoresis
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through a 0.7% agarose gel, using 1X TAE buffer, and comparison to a HindIII DNA
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ladder (New England Biolabs, Ipswich, MA). DNA was stained with ethidium bromide
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and visualized with a UVP MultiDoc-It Gel Imaging system (UVP, Upland, CA).
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PCR amplification and cloning of 16S rRNA genes
DNA extraction was followed by PCR using bacteria-specific primers 27F and
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1492R, based on E. coli numbering (Lane, 1991). PCR amplifications were prepared
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with 1X Buffer, 0.2 mM each dNTP, 1.5 mM MgCl2 (all from Promega), 0.5 µM of each
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primer (Integrated DNA Technologies, Coralville, IA), 1.5 U Taq polymerase (New
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England Biolabs), and ~40 ng DNA template in a final volume of 30 µL. Reactions were
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performed in a TGradient thermal cycler (Whatman Biometra, Goettingen, Germany).
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Amplification of 16S rRNA genes from bacteria consisted of 30 cycles of 95C for 30
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sec, 56C for 30 sec, and 72C for 30 sec with an initial denaturation at 95C for 5
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minutes and a final extension at 72C for 5 minutes.
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PCR products were purified with the QIAquick PCR purification kit (Qiagen,
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Valencia, CA), and were quantified by comparison to a 1 Kb DNA ladder (New England
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Biolabs) when run on a 1% agarose gel. Cloning of mixed community 16S rRNA gene
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fragments was performed using the pGEM T-easy vector (Promega, Madison, WI).
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Ligation into the vector at an insert to vector ratio of 1:1 and transformation using JM109
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competent cells followed manufacturer’s instructions.
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Sequencing and phylogenetic analysis
From 32 clones, PCR using M13 vector-specific primers was performed. DNA
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sequencing reactions were prepared using the forward primer 27f and were sent to the
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Yale University DNA Analysis Facility. Sequences were edited using the software
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FinchTV (http://www.geospiza.com/Products/finchtv.shtml). Good quality sequences
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were used, for a total of 23 clone DNA sequences. Ribosomal RNA gene sequences were
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searched against the Ribosomal Database Project release 10 (Cole et al., 2009) to
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determine nearest matches. Multiple sequence alignments were created using the
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ClustalW interface within the software package BioEdit (Hall, 1999). BioEdit was used
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to create similarity matrices for all sequences and to determine phylotype groups (based
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on ≥ 97% similarity). Phylogenetic trees were created with MEGA 5 (Tamura et al.,
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2007), using the minimum evolution method with 1000 bootstrap replicates. Possible
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chimeras were detected using the Bellerophon server (Huber et al., 2004) and were
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removed from the analyses.
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Overall, microbial diversity at this site seemed to be very high; in an assessment
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of a small subset of 24 clones, none of the sequences were highly similar (>97%) to each
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other, suggesting that many clones would need to be sampled to gain a full understanding
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of the diversity within this mat community. The bacteria library, when compared to 16S
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rRNA gene sequences in the Ribosomal Database Project (Cole et al., 2009), was not
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clearly dominated by any one phylogenetic group, and few sequences matched strongly
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with any sequences in the database, despite appearing to be good quality sequences.
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From the sequence identity matrix generated in BioEdit, aligned and trimmed sequences
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could be compared to each other and to those selected from the Ribosomal Database.
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One sequence, clone FW quarry S1, had a 97.5% match with an uncultured
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Acidobacterium involved in bioremediation of aliphatic hydrocarbons in contaminated
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soil (Militon et al., 2010). Another clone, L10, matched 97.5% with an uncultured beta
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proteobacterium from an iron oxidation biofilm in a lagoon in Japan (Sakurai et al.,
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unpublished). Clone L7 matched 98.7% with an uncultured beta proteobacterium from
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sediments and plankton of a suboxic freshwater pond (Briee et al., 2007). Clone S10
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matched 98% with an uncultured delta proteobacterium from that same environment
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(Briee, et al., 2007). Clone S3 matched 99.5% with a deltaproteobacteria sequence from
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a redox gradient in Baltic Sea sediment (Edlund et al., 2008). Others matched less
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strongly with Cyanobacteria, Planctomycetes, Geobacter, Verrucomicrobia, or
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Acidobacteria. S14 matched 81.5% with Sphingobacteria from high mountain lake
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epilithic biofilms (Bartrons, unpublished).
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Nucleotide sequence accession numbers
Partial sequences of ~620 bp were deposited in GenBank (Benson et al., 2011),
accession numbers are given in Supplementary Figure 1.
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REFERENCES
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BARTRONS, M. Bacterial diversity from high mountain lake epilithic biofilms.
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Unpublished.
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BENSON D.A., KARSCH-MIZRACHI I., LIPMAN D.J., OSTELL J., and SAYERS E.W. 2011.
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GenBank: Nucleic Acids Research, v. 39, D32-7.
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BRIEE, C., MOREIRA, D., and LOPEZ-GARCIA, P. 2007. Archaeal and bacterial community
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composition of sediment and plankton from a suboxic freshwater pond: Res. Microbiol.
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v. 158, p. 213-227.
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COLE, J.R., WANG, Q., CARDENAS, E., FISH, J., CHAI, B., FARRIS, R.J., KULAM-SYED-
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MOHIDEEN, A.S., MCGARRELL, D.M., MARSH, T., GARRITY, G.M., AND TIEDJE, J.M. 2009
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The Ribosomal Database Project: improved alignments and new tools for rRNA analysis:
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Nucleic Acids Res., v. 37 (Database issue), D141-D145.
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EDLUND, A., HARDEMAN, F., JANSSON, J.K., and SJOLING, S., 2008, Active bacterial
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community structure along vertical redox gradients in Baltic Sea sediment: Environ.
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Microbiol., v. 10, p. 2051-2063.
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HALL, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and
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analysis program for Windows: Nucleic Acids Symposium Series, v. 41, p. 95-98.
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HUBER, T., FAULKNER, G. AND HUGENHOLTZ, P. 2004. Bellerophon: a program to detect
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chimeric sequences in multiple sequence alignments: Bioinformatics, v. 20, p. 2317-
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2319.
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LANE, D.J. 1991. 16S/23S rRNA Sequencing: Nucleic Acid Techniques in Bacterial
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Systematics, in, Stackebrandt, E. and Goodfellow, M., (eds), pp. 115-175. John Wiley
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and Sons, New York.
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MILITON C., BOUCHER D., VACHELARD C., PERCHET G., BARRA V., TROQUET, J.,
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PEYRETAILLADE E., and PEYRET, P., 2010, Bacterial community changes during
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bioremediation of aliphatic hydrocarbon-contaminated soil: FEMS Microbiology
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Ecology, v. 74, p. 669-681.
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SAKURAI, K., TAZAKI, K., and YAMAGUCHI, K. Identification of bacteria in an iron-
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oxidation biofilm at Shibayama lagoon, Ishikawa, Japan. Unpublished.
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TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M., AND KUMAR, S. 2011.
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MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood,
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Evolutionary Distance, and Maximum Parsimony Methods: Molecular Biology and
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Evolution, v. 28, p. 2731-2739.