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Supplementary Information: A rare SAR11 fosmid clone confirming genetic variability in the “Candidatus Pelagibacter ubique” genome Jack A Gilbert*, Martin Mühling and Ian Joint Methods Thirty litres of seawater were collected from the surface of the Western Channel Observatory (WCO: http://www.westernchannelobservatory.org.uk/) site L4 (50.25°N: 04.212°W) on 30 November 2005. SAR11 has previously been found at this site (Mary et al., 2006). The water was pre-filtered through a 1.6 µm GF/A filter (Whatman), followed by filtration through a 0.22 µm Sterivex cartridge (Millipore) which was frozen in liquid nitrogen and stored at -80 °C. DNA was extracted and isolated using the method of Neufeld et al. (2007). A fosmid clone library consisting of 10,000 clones was constructed and screened for the presence of SAR11-like 16S rRNA gene containing clones using SAR11-specific 16S rRNA gene primers (Béjà et al. 2000). The fosmid clone library was constructed using the EpiFOS™ kit (Epicentre). Fosmid DNA was extracted using the FosPrep™ 96 fosmid prep kit (Edge Biosystems) and screened using SAR11-specific 16S rRNA gene primers (Béjà et al. 2000) PCR-based screening for SAR11-like 16S rRNA genes: 25 µl volume with final concentrations of 1x GoTaq Flexi Buffer (Promega), 200 µM of each dNTP (Invitrogen), 2 mM MgCl2, 5 µM each of 433F (CTC TTT CGT GGG GAA GAA A) and 567R (CCA CCT ACG WGC TCT TTA AGC) (Béjà et al. 2000) and 1.25 U of GoTaq DNA Polymerase (Promega). Reaction conditions were as follows: 96 °C for 1 min followed by 30 cycles of 96 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, followed by 72 °C for 5 min. Sequencing and assembly of fosmid clones was carried out by the Edinburgh Sequencing Facility (ESF), part of the NERC (National Environmental Research Council) sponsored Molecular Genetics Facilities. Sequence annotation was performed using YAMAP (http://www.genomics.ceh.ac.uk/yamap/), developed as part of the Marine Metagenomics NERC initiative at the National Environmental Bioinformatic Centre (NEBC). The sequence was deposited with Genbank under the accession number EU410957. Phylogenetic analysis The 16S rDNA sequence from the fosmid clone was first compared to sequences stored in the NCBI sequence database using the BLAST algorithm. Subsequently, the sequence of fosmid clone 01-003783 and sequences with high sequence similarity were imported into the ARB software program (http://www.mikro.biologie.tumuenchen.de/pub/ARB/) and aligned to other Alphaproteobacteria 16S rDNA sequences using the automated alignment tool within ARB (Ludwig et al., 2004). The alignment was further corrected ‘by eye’ taking the secondary structure prediction of the ARB program into account. Additionally, 16S rRNA gene sequences (ca. 1325 bp) were added to the alignment from 26 microorganisms that showed high sequence similarity, based on BLAST searches with the nucleotide sequences of the functional genes found in the fosmid clone. Calculation of the phylogenetic trees was performed using the maximum-parsimony algorithm and the neighbor-joining method with a Jukes-Cantor model and a maximum frequency filter in ARB. A consensus tree was obtained by calculating 100 individual trees with each of the two algorithms and depicting the tree with the most frequent branching. To obtain an even more robust tree a “second-stage consensus tree” was constructed by collapsing those branching points within the neighbor-joining tree that were not supported by the maximumparsimony analysis; this was done applying a strict consensus rule until the branching was supported in both the analyses. Phylogenetic and molecular evolutionary analyses for the HVR2 CDSs with Cand. P. ubique homologues (Supplementary figure 1) were conducted using MEGA version 4 (Tamura et al. 2007). Closely related sequences were selected from the BLASTp analysis of the protein sequence of each CDS. These were imported into a CLUSTALW alignment. Sequences were first automatically aligned and then manually curated and where appropriate were masked to ensure an accurate alignment. The number of sequences chosen for alignment to each CDS varied between 10-20 sequences, including the CDS in question and the Cand. P. ubique homologue. As much as possible similar sequences were included for each CDS to enable inter-comparison between each phylogenetic tree. The tree was calculated using the neighbor-joining method with a Poisson correction model and a 500 replicate bootstrap analysis. A bootstrap consensus tree was calculated and average bootstrap values were included on the trees. References: Béjà O, Suzuki MT, Koonin EV, Aravind L, Hadd A, Nguyen LP et al. (2000). Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ Microbiol 2: 516-529. Brown MV, Schwalbach MS, Hewson I, Fuhrman JA (2005) Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series. Environ. Microbiol. 7: 1466-1479. García-Martínez J, Rodríguez-Valera F (2000) Microdiversity of uncultured marine prokaryotes: the SAR11 cluster and the marine Archaea of Group I. Mol. Ecol. 9: 935-948. 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: 13631371. Mary I, Cummings DG, Biegala IC, Burkhill PH, Archer SD, Zubkov MV. 2006a. Seasonal dynamics of bacterioplankton community structure at a coastal station in the western English Channel. Aquatic Microbial Ecology 42: 119-126. Neufeld, J.D., Schafer, H., Cox, M.J., Boden, R., McDonald, I.R., and Murrell, J.C. (2007) Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism. ISME J 1: 480-491. Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599. Legend to Supplementary Fig. 1: Phylogenetic analysis of the16S rRNA gene sequence of fosmid clone 01-003783. The phylogenetic tree was calculated using ARB (Ludwig et al. 2004). The confidence of branch points was determined by two separate analyses (maximumparsimony, neighbor-joining), with multifurcations indicating branch points that were collapsed using a strict consensus rule until supported in both analyses. The neighborjoining tree was chosen to depict the phylogenic relationship of this “second-stage consensus” tree. Values of 100 bootstrap replicates are indicated in brackets (calculated using the maximum-parsimony / neighbor-joining method) at branching points, but those < 75 are omitted. 16S rRNA gene sequences from ‘uncultured marine bacteria’ (i.e. U75254, U75255, U75257 and DQ009201, DQ009251) are described by Brown et al. (2005) or García-Martínez and Rodríguez-Valera (2000), respectively, or unpublished (AY458635). Legend to Supplementary Fig. 2: Phylogenetic tree analysis of each of the 11 CDS found within “hyper-variable region 2” which had a homologue in the “Candidatus Pelagibacter ubique” genome. Each CDS was aligned against the 10 to 20 most significant hits from the BLASTp analysis and the “Cand. P. ubique homologue. The evolutionary history was inferred using the neighbor-joining method, with bootstrap consensus from 500 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. (A) CDS 2; (B) CDS 3; (C) CDS 4; (D) CDS 7; (E) CDS 8; (F) CDS 9; (G) CDS 12; (H) CDS 17; (I) CDS 18; (J) CDS 21; (K) CDS 22.