Supplementary material
Methods
Bacterial isolation and identification
Seawater was collected during a Western Pacific cruise in July 2011. Aliquots
(200 L) of sea water were spread onto marine agar 2216E (Difco) plates
supplemented with 1.5% Agar. Morphologically different colonies were selected,
inoculated into 4 mL of 2216E medium and incubated at 28 C for 3 days with
agitation at 200 rpm. Cell growth was monitored by measuring the optical density at
600 nm (OD 600) using a spectrophotometer (Spectra max plus, Molecular Devices,
Sunnyvale, CA).
For screening of EPS-producing bacteria, the supernatant was collected from the
culture medium using centrifugation at 8,000 g for 5 min. The content of the EPS in
the supernatant was measured using the phenol/H2SO4 method [1] using glucose as
the standard. The isolated strains were identified using 16S rRNA gene sequence
analysis. The genomic DNA was extracted from cells grown in 2216E medium
overnight at 28C using the method described by Marmur [2]. A 16S rRNA gene was
amplified by means of a polymerase chain reaction (PCR) using genomic DNA as a
template, and the bacterial universal primers corresponding to E. coli position 8F
(5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGT TAC CTT GTT ACG
ACT T-3’). PCR thermal conditions were as follows: 94 C for 3 min, 30 cycles of
denaturation at 94 C for 30 s, annealing at 55 C for 1 min, extension at 72 C for 2
min, and a final extension at 72 C for 7 min. An amplification product of about
1.5-kb was purified and sequenced. The 16S rRNA gene sequence was compared to
the sequences of the most closely related strains in the GenBank database and
EzTaxon server 2.1 [3]. For phylogenetic analysis, the 16S rRNA gene sequences of
closely related, validly published taxa were retrieved from the GenBank database
using
BLAST,
and
aligned
using
CLUSTAL
X
(ftp://ftp.ebi.ac.uk/pub/software/clustalw2). A phylogenetic tree was constructed
using MEGA 4 [4]. Bootstrap analysis of 1000 replications was performed to assess
the confidence limits of the branching.
Transmission electron microscopy observations
JL2069 cells grown on 2216E solid medium at 28 C for 24 h were used for
preparing transmission electron microscopy (TEM) samples. Cells collected from the
medium were either directly immersed in 2% phosphotungstic acid (pH 7.0) or rinsed
twice with 1mL of 0.85% NaCl before being immersed in 2% phosphotungstic acid.
The cells were then loaded onto a copper grid and dried at room temperature.
Observations were made under standard conditions with a JEM-1230 (JEOL Ltd.
Tokyo, Japan)
Figures
Fig. 1. Neighbor-joining tree showing the phylogenetic position of strain JL2069 and
representatives of other taxa based on 16S rDNA sequences. Bar = 0.01 substitutions
per nucleotide position. Bootstrap values expressed as percentages of 1000 replicates
greater than 50% are shown at the branch points.
Fig. 2. Electron micrograph of negatively stained cells of JL2069. The cells cultured
on the 2216E solid medium for 24 h at 28 C were collected and directly stained with
2% phosphotungstic acid (A) or washed three times by 0.85% NaCl before staining
(B). Scale bar = 1m.
References
1. Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F (1956) Colorimetric method
for determination of sugars and related substances. Anal Chem 28: 350-356
2. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from
microorganisms. J Mol Biol 3: 208-218
3. Chun J, Lee JH, Jung Y, Kim M, Kim S, et al. (2007) EzTaxon: a web-based tool
for the identification of prokaryotes based on 16S ribosomal RNA gene
sequences. Int J Syst Evol Microbiol 57: 2259-2261
4. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software
for evolutionary analysis of DNA and protein sequences. Briefings in
bioinformatics 9: 299-306