SUPPLEMENTARY MATERIALS AND METHODS
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Growth and stress conditions. Succinate 0.5% (wt/v) or benzoate 4 mM was added to the
MMM as a source of energy and carbon. Yeast extract was added at a final 0.005% (wt/v) to
MMM as a source of vitamins and growth factors. If not specifically mentioned, cells were
incubated at 26 °C under 180 rpm agitation. Exponential phase was considered when culture
OD600 was in the 0.6-0.7 range. Early stationary phase was considered when culture reached
an OD600 of 1.2-1.3 in marine broth under optimal conditions, whereas mid and late stationary
phases were considered 24 and 72 h after this point, respectively. Microanaerobic conditions
were obtained by incubating the medium in bottles completely filled, i.e. without air. These
conditions led to very slow and poor growth (OD600 = 0.4). For UV stress, cell irradiation at
254 nm took place at 1.85 J/sec per 100-ml cultures. After UV stress or temperature shock (42
ºC) was applied, cultures were incubated under standard conditions for 30 min. Cells referred
to as MB-harvested cells in Table 1 and used for diverse incubations were obtained from an
optimal marine broth culture grown to the early stationary phase and harvested by
centrifugation at 3,000 x g at 20 °C for 10 min. Filter sterilised naphthalene (Sigma) solution
at 0.5% (wt/v) and commercial diesel (0.03, 0.3 and 3% v/v) were added to marine brothcontaining flasks prior to inoculum. Sea-water was obtained from an open beach (La GrandeMotte, coordinates 43.55N/4.03E, France, 26th of October 2009) and autoclaved prior to use.
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30
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50
Protein extracts and SDS-PAGE. Cell pellets were resuspended in 5 volumes of P buffer
consisting in 100mM sodium phosphate buffered at pH 8.2 and containing Complete Protease
Inhibitor (1 tablet per 42 ml, Roche). Cells were lysed at 4°C by sonication applying a 40 J
dose with amplitude of vibration of 20% and pulses of 5 seconds followed by resting intervals
of 5 seconds. Lysates were centrifuged for 20 min at 14,000 g at 4°C to remove cellular
debris. Protein content from the soluble fractions was quantified by the Bradford based BioRad protein assay kit (BioRad). Lithium dodecyl sulphate-β-mercaptoethanol (LDS) protein
gel sample buffer (Invitrogen) was added to the protein fractions. The resulting samples were
incubated at 99°C for 5 min prior SDS-PAGE. A protein quantity of 50 and 100 μg were
loaded on a 10% Tris-Bis NuPAGE gel (Invitrogen) for short and long electrophoresis
migrations,
respectively.
SDS-PAGE
was
carried
out
using
1X
3-(Nmorpholino)propanesulfonic acid solution (Invitrogen) as the running buffer. For the one
nanoLC-MS/MS run shotgun analysis, proteins were resolved only over 3 mm by means of a
short migration, whereas migration was allowed over 5 cm for the 15-band analysis. Gels
were stained with SimplyBlue SafeStain, a ready-to-use Coomassie G-250 stain from
Invitrogen. SeeBlue Plus2 (Invitrogen) was used as molecular weight marker.
Subproteome fractioning. To obtain the membrane fraction, the pellet of cell debris obtained
after cell sonication and centrifugation was resuspended in 1 ml of phosphate buffer. This
sample was centrifuged for 2 min at 6,000 x g to eliminate any remaining intact cells. The
resulting supernatant was centrifuged for 30 min at 13,000 x g. The pelleted membrane
fraction was washed twice with phosphate buffer in order to eliminate cytosolic contaminant
proteins. The final pellet was resuspended in 20 μl of LDS protein gel sample buffer. The
sample was then incubated at 99°C for 5 min prior to short SDS-PAGE migration.
Phosphocellulose P11 chromatography was carried out to enrich proteins with affinity for
phosphate groups as described by Gauci and co-workers (Gauci et al 2009). For this, cell
pellets were resuspended in phosphocellulose buffer (PH buffer) consisting in 20 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 8.0, 0.01% Tween, 10%
glycerol, 75 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF and Roche Complete
Protease Inhibitor (6 tablets per 250 ml), and sonicated. The soluble fractions were desalted
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60
65
70
75
80
85
90
95
100
using two coupled HiTrap (5ml) desalting columns (GE Healthcare) operated with an Äkta
Purifier 10 FPLC system at a flow rate of 1 ml/min. The proteins were eluted from the column
with PH buffer. Phosphocellulose P11 chromatography was carried out with an HR 10 x 5
column (Amersham Biosciences) packed with 2 ml of P11 fibrous cellulose phosphate cationexchanger gel (Whatman). The gel was equilibrated with PH buffer. Desalted protein extract
(2 mg) was applied to the gel. Proteins were eluted by an increasing step gradient of NaCl:
0.1, 0.3, 0.5, 0.85, and 1.2 M. They were concentrated by trichloroacetic acid precipitation as
described previously (Christie-Oleza and Armengaud 2010). The resulting pellets were
resuspended in 60 μl LDS protein gel sample buffer and incubated at 99°C for 5 min prior
SDS-PAGE. A volume of 10 μl of each sample was loaded on the SDS-PAGE gel for short
electrophoretic migration. Proteins in 0.1 and 1.2 M NaCl fractions were mixed with 0.3 and
0.85 M NaCl fractions, respectively. Phosphocellulose-enriched proteins from each of the five
conditions assayed resulted in three samples analysed by one nanoLC-MS/MS run each.
NanoLC-MS/MS, MS/MS database search and spectral count. For MS/MS database
searches, a protein sequence database containing all the annotated protein coding sequences
(CDS) of R. pomeroyi DSS-3 genome (Moran et al 2004), i.e. chromosome (NC_003911) and
pDSS-3 megaplasmid (NC_006569) deposited at the NCBI, was made in-house. Peak lists
were generated with Matrix Science MASCOT DAEMON software (version 2.2.2) using the
extract_msn.exe data import filter (ThermoFisher) from the ThermoFisher Xcalibur FT
package (version 2.0.7). Data import filter options were set at: 400 (minimum mass), 5,000
(maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1,000 (threshold).
MS/MS spectra were searched against the in-house database with the MASCOT 2.2.04
software (Matrix Science). Search parameters were as previously described (Christie-Oleza
and Armengaud 2010) but allowing 2 miss-cleavages. MASCOT results were parsed using
the IRMa 1.22.4 software (Dupierris et al 2009) filtering with a p value below 0.02. A protein
was considered valid when at least two different peptides were detected.
False positive rate calculation. When merging all the data presented in this manuscript, a
global false positive rate of 1.2% for protein identification was estimated using a reverse
decoy database. This was performed by calculating the spectral assignments of the compiled
MS/MS data against the reverse sequences present in the R. pomeroyi CDS database (decoy
database). Possible peptide attributions to the decoy database are just by chance.
Hierarchical clustering and protein discovery saturation curves. Hierarchical clustering of
the 30 conditions was performed using the corresponding polypeptides normalised spectral
abundance factor values and the cluster analysis option of the PAST v.2.01 analysis package
(Hammer et al 2001). Default parameters were used for unweighted pair-group average
(UPGMA) algorithm and the Euclidean, Correlation, Cosine and Morisita’s index distance
calculation. Bootstrap values were calculated over 1,000 tree replicates. Protein saturation
curves were produced using the sample rarefaction option in the PAST v.2.01 package.
Extrapolation of the curves was performed with logarithmic and powered algorithms.
Protein sequence analysis. CDS from R. pomeroyi homologous to the 382 proteins specified
as essential genes for Mycoplasma genitalium by Glass and co-workers (Glass et al 2006)
were searched with the BioEdit BLASTP tool v.7.0.5 (Hall 1999) and further manual
inspection. BLASTP searches were carried out for establishing the Roseobacter core
proteome against the 32 genomes currently present in the Roseobase database
(www.roseobase.org). These were: Dinoroseobacter shibae DFL 12, Roseobacter sp. AzwK3b, Roseovarius nubinhibens ISM, Jannaschia sp. CCS1, Roseobacter denitrificans OCh 114,
2
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110
Roseovarius TM1035, Loktanella vestfoldensis SKA53, Roseobacter sp. CCS2, Sagittula
stellata E-37, Oceanicola batsensis HTCC2597, Roseobacter sp. MED193, Ruegeria
pomeroyi DSS-3, Oceanicola granulosus HTCC2516, Silicibacter sp. TM1040,
Rhodobacterales HTCC2255, Roseobacter SK209-2-6, Sulfitobacter sp. EE-36,
Rhodobacterales HTCC2654, Roseovarius sp. 217, Sulfitobacter NAS-14.1, Phaeobacter
gallaeciensis BS107, Roseobacter litoralis Och149, Roseovarius sp. HTCC2601,
Oceanibulbus indolifex HEL-45, Roseobacter HTCC2150, Octadecabacter antarcticus str
238, Octadecabacter antarcticus str 307, Phaeobacter gallaeciensis 2.10, Rhodobacterales
HTCC2083, Rhodobacterales Y4I, Roseobacter GAI101 and Ruegeria sp R11. Only
homologue identifications with E values below 10e-20 were considered.
References.
115
Christie-Oleza JA, Armengaud J (2010). In-depth analysis of exoproteomes from marine
bacteria by shotgun liquid chromatography-tandem mass spectrometry: the Ruegeria
pomeroyi DSS-3 case-study. Mar Drugs 8: 2223-2239.
120
125
Dupierris V, Masselon C, Court M, Kieffer-Jaquinod S, Bruley C (2009). A toolbox for
validation of mass spectrometry peptides identification and generation of database: IRMa.
Bioinformatics 25: 1980-1981.
Gauci S, Veenhoff LM, Heck AJ, Krijgsveld J (2009). Orthogonal separation techniques for
the characterization of the yeast nuclear proteome. J Proteome Res 8: 3451-3463.
Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M et al (2006).
Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A 103: 425-430.
130
Hall TA (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95-98.
Hammer Ø, Harper DAT, Ryan PD (2001). PAST: Paleontological statistics software package
for education and data anlysis. Palaeont Electronica 4: 1-9.
135
Moran MA, Buchan A, Gonzalez JM, Heidelberg JF, Whitman WB, Kiene RP et al (2004).
Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment.
Nature 432: 910-913.
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