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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.