|Supplementary information: Materials and Methods
Cultivation of Cyanobacteria from Elkhorn Slough Microbial Mats
Mat material was transferred onto ASN- or on modified ASN agarose plates (ESFC-1 was initially enriched on ASN- plates). Cultures were subsequently further isolated and maintained on a modified ASN liquid medium with nitrate to yield more biomass and to maintain the cultures. Cultures were grown on dark-light cycle (12h/12h) at 22 C with a light intensity of ca. 40 µmol photons m-2 s-1.
Composition of modified ASN or ASN- medium (no NaNO3 added) (modified by Leslie Prufert-Bebout after the ASN-III medium (Rippka, 1988))
Salts: 427 mM NaCl; 9.8 mM MgCl2; 6.7 mM KCl; 14.2 mM MgSO4; 3.4 mM CaCl2; 2.4 mM NaHCO3; 0.14 mM citric acid
Basic Nutrients: 36.3 µM KH2PO4; 11.7 µM FeCl3; 5.93x10-7 M thiamine HCl (vitamin B1); 4x10-9 M cyanocobalamin (vitamin B12); 2x10-8 M biotin (vitamin H) and 1 ml/l of “A-5 + Co” trace metal solution without CuSO4 after (Rippka, 1988)
Optional Nutrients: 0.88 mM NaNO3
Adjust pH to 7.8 to 8.2 and salinity to 28‰. After autoclaving and cooling add 1 ml of vitamin stock solution.
DNA and RNA Extraction
RNA and DNA were co-extracted from the uppermost 2 mm of 3 pooled mat cores by combining phenol-chloroform extraction with parts of the RNeasyMini and QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA), respectively. For each core (10 mm diameter, upper 2 mm), biomass was transferred to a tube contaning 0.5 ml RLTTM buffer and homogenized using a rotor-stator homogenizer (Omni International, Kennesaw, GA, USA). The suspension was bead-beated (BioSpec Products, Bartlesville, OK, USA) with zirconium beads (200 µm, OPS Diagnostics, Lebanon, USA) and centrifuged at 8,000 x g for 1 minute. Supernatants from 3 mat cores were pooled and split into two aliquots; one for RNA and the other for DNA extraction. Each aliquot was extracted with phenol-chloroform-isoamyl alcohol (125:24:1, pH 4.5 for RNA and 25:24:1, pH 8.0 for DNA extraction) and the aqueous phase was further purified following the manufacturers’ protocols. For the RNA samples, the aqueous phase was run through the gDNA eliminator spin column (QIAGEN) to remove genomic DNA and further treated with TURBO DNaseTM (Applied Biosystems/Ambion, Austin, TX, USA). Two µg of isolated RNA were reverse transcribed into single-stranded cDNA using the SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). For the cyanobacterial culture ESFC-1, ca. 300 mg of biomass was transferred into 0.5 ml RLT TM buffer, treated by bead-beating, and DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN) as mentioned above.
Construction of 16S rRNA Gene/Transcripts and NifH Gene/Transcript Clone Libraries
16S rRNA gene sequences from the cyanobacterial culture ESFC-1 were amplified using the general bacterial primers 27F (Lane, 1991) and 1391R (Lane, 1991). Positive PCR products from five reactions were pooled and purified using the Wizard SV Gel and PCR Clean-up system (Promega, Madison, WI, USA). Purified PCR amplicons were cloned using the TOPO TA Cloning Kit for sequencing (Invitrogen) according to the manufacturer’s protocol. Clones were screened with cyanobacteria specific primers Cya359F and Cya781(a and b) R targeting the cyanobacterial 16S rRNA gene (Nübel et al., 1997). Cyanobacterial positive clones were sequenced by Sequetech (Mountain View, CA, USA).
General 16S rRNA clone libraries of Elkhorn Slough mat were generated from cDNA samples extracted from mat samples collected in 12th/13th January 2009 (12th of January, 9:00 pm and 13th of January, 7:00 am) from the upper 2 mm of the mat. 16S rRNA genes were amplified with primers 27F and 1391R and clone libraries were constructed and sequenced at the Department of Energy Joint Genome Institute (JGI, Walnut Creek, CA, USA) according to standard protocols (http://my.jgi.doe.gov/general/index.html).
Clone libraries of the dinitrogenase reductase gene (nifH) were constructed from mat cores sampled during two consecutive diels: 21st October 2009, at 10:50 pm and 24th October 2009, at 3:10 am. DNA and RNA was extracted from the uppermost 2 mm and RNA was reverse transcribed into cDNA as decribed above. The nifH genes were PCR amplified (in 5 replicates) from DNA and cDNA following the published protocol from (Zehr and Turner, 2001) of a nested PCR. The primer sites were described as being conserved throughout nifH genes in clusters I, II, III and IV. The first PCR was conducted with primers nifH4 and nifH3, followed by a second PCR reaction with primers nifH1 and nifH2. The PCR products were approximately 359 bp in length. Amplicons were gel purified, ligated and transformed as mentioned above. Clones were screened for insert size and positive clones were sequenced by Sequetech (Mountain View, CA, USA).
Construction of 16S rRNA Gene and Transcript 454 Pyrotag Libraries
Amplicons of the 16S rRNA V8 hypervariable region were constructed from 7 time points in the year 2009 (13th January, 30th April, 1st July, 19th August, 16th September, 21stOctober and 13th November 2009) from the uppermost 2 mm of the mats and sequenced at the Department of Energy Joint Genome Institute using previously published protocols (Engelbrektson et al., 2010). The V8 region of cDNA or DNA was PCR amplified using the universal primers 926F (Lane, 1991) and 1392R (Lane et al., 1985). The reverse primer included the adaptor sequence and a five-base barcode.
16S rRNA genes. 16S rRNA gene/transcript sequences were quality checked and nearly full-length sequences were assembled using Geneious 5.0.3 (http://www.geneious.com). Sequences were aligned using the SILVA website (http://www.arb-silva.de) and imported in the ARB program (Ludwig et al., 2004) with the SILVA 94 Ref database, supplemented with cyanobacterial sequences from SILVA Ref 104. Phylogenetic trees were calculated using maximum likelihood, maximum parsimony and neighbor joining algorithms, with and without a 50% position variablity filter. Bootstrap values were calculated in Geneious 5.0.3 with the PhyML algorithm, using 100 bootstrap trees.
NifH genes. NifH gene/transcript sequences were quality checked in Geneious. Deduced amino acid sequences of the nifH genes/expressed genes along with closest related nifH sequences collected from NCBI and reference sequences from the Zehr laboratory nifH database (http://www.es.ucsc.edu/~wwwzehr/research/database) were locally aligned in MUSCLE (Edgar, 2004) and imported into the ARB program. Phylogenetic trees were determined based on deduced amino acid sequences using maximum likelihood, maximum parsimony and neighbor joining algorithms. Bootstrap values were calculated as described above. Representative chlorophillide reductase genes were included in the nifH database to detect sequences related to those genes in the sample. No sequences from the investigated samples grouped with those representative genes.
454 Pyrotag Amplicons of the V8 region. Pyrosequencing amplicons were quality checked using the RDP pipeline with standard settings (http://pyro.cme.msu.edu). After quality assessment, the time point “13th January; 7:00 am” had a total of 20,068 sequences of DNA and 9,338 sequences of cDNA samples, the time point “30th April; 13:10 pm” had a total of 11,447 DNA and 7,303 cDNA sequences, the time point “1st July; 4:00 am” a total of 15,403 DNA and 8,711 cDNA sequences, the time point “19th August; 1:15 am” a total of 6,216 DNA and 10,532 cDNA sequences, the time point “16th September; 12:00 pm” a total of 8,088 DNA sequences, the time point “21st October; 10:50 pm” a total of 17,941 DNA and 5,586 cDNA sequences, and the time point “13th November; 12:00 am” a total of 6,354 DNA and 5,032 cDNA sequences. Pyrotag data were screened for reads affiliated with ESFC-1 in Geneious 5.0.3 by conducting a MegaBLAST search of the ESFC-1 16S rRNA sequence (nearly full length) against the pyrotag reads. Based on testing the sequence identity of the V8 region from the ESFC-1 enrichment culture against cyanobacterial sequences outside of the ESFC-1 cluster (max. sequence identity 96.2%) in ARB, the prerequisite for a pyrotag being assigned as ESFC-1 affiliated were set as follows: sequence alignment with the 16S rRNA sequence of ESFC-1 of at least 400 bp and a sequence identity of 96.5%. The proportions of ESFC-1 affiliated pyrotag reads to the total number of pyrotag reads with a length 400bp are displayed in Figure 5.
Design and Optimization of ESFC-1-Specific Oligonucleotide Probes
The ProbeDesign tool in ARB was used to design FISH probes specific for the monophyletic ESFC-1 cluster. The specificity of potential probe sequences was examined using probeCheck (Loy et al., 2008), the probe Match function in RDP (http://rdp.cme.msu.edu/probematch/search.jsp) and ARB (using the SSU_102_ref database). Based on these examinations, two probe sequences were chosen for optimization: ESFC1_172 and ESFC1_177 (Supplementary Table 1). To optimize the hybridization conditions of these probes, cyanobacterial strains with the lowest mismatch case for each probe were chosen (Supplementary Table 1): Halospirulina tapeticola, strain CCC Baja-95 Cl.2 (CCMEE number 5550) and Oscillatoria sp. Ant-Salt (CCMEE number 5011). Both cultures were provided by the Culture Collection of Microorganisms from Extreme Environments (CCMEE, Eugene, OR, USA). Those cyanobacterial strains were grown in BG11 (Stanier et al., 1971) media at 22 C and 12 C (respectively) and fixed in ethanol (Schönhuber et al., 1999). For CARD-FISH optimization, these cyanobacterial cultures and ESFC-1 were spotted onto VectaBond (Vector Laboratories, Burlingame, CA, USA) coated slides and attached by drying. Oscillatoria sp. and Halospirulina tapeticola were hybridized with the eubacterial probe EUB I-III (Amann et al., 1990; Daims et al., 1999) serving as the control for maximal hybridization intensity. Cultures were hybridized with probes ESFC1_172 and ESFC1_177 with increasing formamide concentrations and stained with 4',6-diamidino-2-phenylindole (DAPI). Cells were imaged on an Axioplan epifluorescence microscope (Carl Zeiss, Jena, Germany) with the Axiovision program and pictures of CARD-FISH stained cells were taken with consistent exposure times and settings. The signal intensities were measured with ImageJ (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institute of Health, Bethesda, MD, USA).
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. (1990). Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56: 1919-1925.
Daims H, Brühl A, Amann R, Schleifer KH, Wagner M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434-444.
Edgar RC. (2004). MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797.
Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H et al. (2010). Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J 4: 642-647.
Lane DJ. (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds.). Nucleic acid techniques in bacterial systematics. John Wiley and Sons: New York, pp 115–175.
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82: 6955-6959.
Loy A, Arnold R, Tischler P, Rattei T, Wagner M, Horn M. (2008). ProbeCheck - a central resource for evaluating oligonucleotide probe coverage and specificity. Environ Microbiol 10: 2894-2898.
Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004). Arb: A software environment for sequence data. Nucleic Acids Res 32: 1363-1371.
Nübel U, Garcia-Pichel F, Muyzer G. (1997). PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol 63: 3327-3332.
Rippka R. (1988). Isolation and purification of cyanobacteria. Meth Enzymol 167: 3-27.
Schönhuber W, Zarda B, Eix S, Rippka R, Herdman M, Ludwig W et al. (1999). In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 65: 1259-1267.
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35: 171-205.
Zehr, J. P., Turner PJ. (2001). Nitrogen fixation: Nitrogenase genes and gene expression. In: Paul JH (ed.). Methods in Microbiology: Marine Microbiology. Academic Press: San Diego, pp 271–286.