The k.o. strains of hprK kinase (mpn223), prpC (mpn247) phosphatase and pknB (mpn248) kinase were generated by transposon-mediated insertion of a gentamycin resistance cassette in wild-type M. pneumoniae
M129 (ATTC29342 broth passage No. 31) (Halbedel et al, 2006
; Schmidl et al, 2010b
). Three 100-ml cultures were inoculated with either wild-type M. pneumoniae
M129 or one of the three k.o. strains, and the cultures were grown in modified Hayflick medium without antibiotics for 96 h until late exponential phase. Wild-type M. pneumoniae
, hprK (mpn223) kinase k.o. strain and prpC (mpn247) phosphatase k.o. strain were then washed three times with ice-cold phosphate-buffered saline (PBS), scraped from the bottom of the flask and centrifuged at 9860 g
. As the pknB (mpn248) k.o. strain does not grow adherent, the cells were centrifuged at 9860 g
and then washed three times with ice-cold PBS. Of wild-type and pknB (mpn248) k.o., three such pellets were generated and two pellets were generated for hprK (mpn223) k.o. and prpC (mpn247) k.o. to serve as biological replicates.
Cell pellets were resuspended in urea buffer (8 M urea (Merck), 50 mM ammonium bicarbonate (Fluka), 1 mM sodium vanadate (Merck), 1 mM potassium fluoride (Fluka), 5 mM sodium phosphate (Sigma), supplemented with protease and phosphatase inhibitors (Roche)) and homogenized using a glass douncer. Cells were then lysed by sonication (6 × 20 s, 40 s pause, 80% output level, 50% duty cycle, using an Ultrasonic processor UIS250v and a VialTweeter of Hielscher Ultrasound Technology) and insoluble debris was pelleted at 10 000 g in a table-top centrifuge (Eppendorf 5415D) at 4°C. Protein concentrations were determined for all lysates by Bradford assay (Bio-Rad) and adjusted to 2.5 mg/ml using urea buffer. Cell lysates were snap frozen at –80°C until further processing.
For the proteomic and PTM analysis, lysates for three biological replicates of wild-type M. pneumoniae, two hprK (mpn223) k.o., two prpC (mpn247) k.o. and three pknB (mpn248) k.o. (indicated as C1–3 in ) were produced as described above. From each lysate, two equivalents of 500 μg protein, each, were further processed as technical duplicates. Cysteines were reduced with 5 mM dithiothreitol (DTT) for 15 min at 56°C and subsequently alkylated with 10 mM iodacetamide (Sigma) for 30 min at 25°C in the dark. Proteomes were then digested with 4 μg endoprotease LysC for 4 h at 37°C and the solutions were then diluted with 50 mM ammonium bicarbonate (Sigma) to a final urea concentration of 1 M. The proteomes were further digested by incubation with 8 μg trypsin protease for additional 18 h at 37°C, followed by an additional incubation with 8 μg trypsin at 37°C for another 5 h.
Differential dimethyl labeling of peptides and combining of proteomes
The resulting peptides were bound to a C18 SepPak column and differentially modified with a dimethyl label on the column following the protocol of Boersema et al (2009)
. ‘Light'-, ‘medium'- and ‘heavy'-labeled peptide solutions were then combined according to the scheme in to give a total of six proteome combinations.
SCX chromatography for peptide fractionation
Each of the six proteome combinations was fractionated using SCX chromatography to separate phosphorylated from unmodified peptides, monitored by UV absorbance.
Peptides from each digest corresponding to 1.5 mg of protein material were loaded onto two C18 cartridges using an Agilent 1100 HPLC system. The flow rate applied was 100 μl/min using water, 0.05% formic acid (FA), pH 2.7, as solvent. Subsequently, peptides were eluted from the trapping cartridges with 80% acetonitrile, 0.05% FA, pH 2.7, onto a PolySULFOETHYL A column 200 × 2.1 mm2 (PolyLCinc.) for 10 min at the same flow rate. Separation was performed using a non-linear 65 min gradient, 0–10 min 100% solvent A (5 mM KH2PO4, 30% Acetonitrile, 0.05% FA, pH 2.7), 10–15 min up to 26% solvent B (5 mM KH2PO4, 30% acetonitrile, 350 mM KCl, 0.05% FA, pH 2.7), 15–40 min to 35% solvent B and from 40 to 45 min to 60% solvent B. At 49 min, the concentration of solvent B was 100%. The column was subsequently washed for 6 min with high salt concentration and finally equilibrated with 100% solvent A for 9 min. The flow rate applied during the SCX gradient was 200 μl/min.
Fractions were collected at 1-min intervals for 40 min. After evaporation of the solvents, fractionated peptides were resuspended in 10% FA. Of each of the fractions 11–22 20% and of the fractions 26–30 0.4% were then analyzed by reversed phase LC-MS/MS.
Enrichment of lysine-acetylated peptides
For the investigation of the acetylome of M. pneumoniae
, one lysate of each strain was analyzed. A cocktail of deacetylase inhibitors (Trichostatin A (10 μM), nicotinamid (10 mM) and butyric acid (50 mM)) was added to the urea lysis buffer mentioned for the phosphoproteomic analysis. Lysis, proteome digestion and peptide labeling were essentially performed as described for the phosphoproteomic analysis above. Acetylated peptides were immunoprecipitated following the protocol published by Choudhary et al (2009)
. In brief, after digestion, dimethyl labeling and proteome combination according to , the equivalent of 5 mg of peptides was lyophilized overnight and subsequently dissolved in acetyl-lysine affinity purification buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM sodium chloride). After incubation with anti-acetyl-lysine affinity resins (ImmuneChem, Canada) for 14 h at 4°C on a rotating wheel, the resins were then washed four times with acetylated-lysine affinity purification buffer and two additional times with distilled water. Acetylated peptides were eluted with 0.1% trifluoroacetic acid (TFA). The eluates and flow throughs were desalted using StageTips as described in Rappsilber et al (2007)
prior to LC-MS/MS analysis.
The analysis of the SCX fractions was performed using a nano LC-LTQ-Orbitrap Classic (Thermo). An Agilent 1200 series LC system was equipped with a 20-mm Aqua C18 (Phenomenex, Torrance, CA) trapping column (packed in-house, i.d., 100 μm; resin, 5 μm) and a 400-mm ReproSil-Pur C18-AQ (Dr Maisch GmbH, Ammerbuch, Germany) analytical column (packed in-house, i.d., 50 μm; resin, 3 μm). Trapping was performed at 5 μl/min for 10 min in solvent A (0.1 M acetic acid in water), and elution was achieved with a gradient of 10–35% B (0.1 M acetic acid in 80/20 acetonitrile/water) in 90 min in a total analysis time of 120 min (fractions 11–22), or in 135 min in a total analysis time of 180 min. The flow rate was passively split to 100 nL/min when performing the elution analysis. Nanospray was achieved using a distally coated fused silica emitter (New Objective, Cambridge, MA) (o.d., 360 μm; i.d., 20 μm, tip i.d. 10 μm) biased to 1.7 kV. A 33-MΩ resistor was introduced between the high voltage supply and the electrospray needle to reduce ion current.
The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full scan MS spectra (300–1500 m/z) were acquired with a resolution of 60 000 at 400 m/z after accumulation to a target value of 500 000. The five (fractions 11–22) or 10 (fractions 26–30) most intense peaks above a threshold of 500 were selected for collision-induced dissociation in the linear ion trap at normalized collision energy of 35% after accumulation to a target value of 30 000.
The acetyl-lysine enriched and depleted peptide mixtures were analyzed by chromatographic separation on a EASY-nLCTM system (Proxeon Biosystems) fitted with a trapping (self-packed Hydro-RP C18 (Phenomenex), 100 μm × 2.5 cm, 4 μm) and an analytical column (self-packed Reprosil C18 (Dr Maisch) 75 μm × 15 cm, 3 μm, 100 Å). The outlet of the analytical column was coupled directly to an LTQ-OrbitrapVelos (Thermo Scientific) using a Thermo Scientific Nanospray Flex Ion Source. Solvent A was water, 0.1% FA and solvent B was acetonitrile, 0.1% FA. The samples (1 μl in 5% acetonitrile, 5% FA) were loaded with a constant flow of solvent A at 20 μl/min onto the trapping column. Trapping time was 1 min. Peptides were eluted via the analytical column at a constant flow of 0.3 μl/min. During the elution step for the acetyl-enriched samples, the percentage of solvent B increased in linear gradients from 5 to 25% B in 40 min, then from 25% B to 80% in 5 min, to a total gradient time of 60 min including a final wash step of 15 min at 80% B. For the elution of the acetyl-lysine-depleted samples, the percentage of solvent B increased in linear gradients from 5 to 25% B in 90 min, then from 25% B to 40% in 10 min and finally from 40 to 80% B in 10 min, to a total gradient time of 120 min including a final wash step of 10 min at 80% B. The peptides were introduced into the mass spectrometer via a Pico-Tip Emitter 360 μm OD × 20 μm ID; 10 μm tip (New Objective), and a spray voltage of 1.9 kV was applied. The capillary temperature was set to 200°C. Full scan MS spectra with a mass range 300–1700 m/z were acquired in profile mode in the FT with a resolution of 30 000. The filling time was set at maximum of 500 ms with limitation of 106 ions. The most intense ions (up to 15) from the full scan MS were selected for sequencing in the LTQ. Normalized collision energy of 40% was used, and the fragmentation was performed after accumulation of 3 × 104 ions or after filling time of 50 ms for each precursor ion (whichever occurred first). MS/MS data were acquired in centroid mode. Only multiply charged (2+ and 3+) precursor ions were selected for MS/MS. The dynamic exclusion list was restricted to 500 entries with maximum retention period of 30 s and relative mass window of 10 p.p.m. In order to improve the mass accuracy, a lock mass correction using a background ion (m/z 445.12003) was applied.
OejVCeG2v3KJap1OlQbfeYM3KvQwv6EtRRw9+msFLnPpLl/4MBuKKp6hdI/ZgX2JNW1pUoUGAUeMro8FRgIOkLp/tW8AAAAAAAAFNg==. Additionally, the scaffold files and raw data are available at http://vm-lux.embl.de/Docu/VanNoortMSB2012/
and from TRANCHE using the hash 8YQSJO0UiPO2JoOgE2DZ3yXolC5cGOCOhra/0kvrMLRGKagf1fXUJ2w1c/5DdbkS9/k0aIDW0d4+qR/Kpz03zrrvCDAAAAAAAABvYw==. Note that scaffold files contain the raw mascot results loaded into the Scaffold tool with some default filter criteria and were not filtered the same way as described in the Materials and methods section, and also the FDR calculations there will be different than in our final data sets.
Peak lists in the Mascot generic text file format were extracted from Thermo raw data files using the Quant application in the MaxQuant environment (Cox and Mann, 2008
) (version 18.104.22.168). Results from all LC-MS/MS experiments of each proteome combination were combined into a single file and analyzed with the Matrix Science Mascot search engine (version 2.2.03) using a UniProt protein database of M. pneumoniae
(downloaded on 18 May 2010 from http://www.uniprot.org
) plus previously identified contaminants. Search parameters were chosen as follows: trypsin as the proteolytic enzyme, up to three missed cleavages, cysteine carbamidomethylation as a fixed modification and methionine oxidation as well as serine/threonine/tyrosine phosphorylation as variable modifications. Instead of searching for differential dimethyl labels as variable modifications (see below), dimethyl (‘light' (12
), ‘medium' (12
) and ‘heavy' (13
)) lysine and peptide N-termini were defined as exclusive modifications in the Mascot-specific ‘Quantitation' mode. The peptide tolerance was set to 15 p.p.m., and the MS/MS tolerance was set to 0.6 Da. The ‘Decoy' option was used for subsequent peptide FDR-based filtering (see below). The data of all SCX fractions of a proteome combination were searched together. Only unique peptides in the protein database were taken into account. For this the following rules to identify peptides were applied: (1) the peptide is unique among SwissProt M. pneumoniae
proteins or (2) the peptide is unique in UniProt (SwissProt + trEMBL) M. pneumoniae
proteins else the peptide identification is discarded.
Modified and unmodified peptide filtering
We used RockerBox (van den Toorn et al, 2011
) to filter all peptides (modified and unmodified) to a 1% peptide FDR, using the Mascot Percolator option (Kall et al, 2007
) for each proteome combination separately (Supplementary Table S2
). For this purpose, we trained RockerBox on the complete set of peptides (including phosphorylated or lysine-acetylated) and then separated the target and decoy peptides into modified and unmodified sets. Two separate score thresholds were set per proteome combination such that both the modified and unmodified peptides had a peptide FDR of 1%. As phosphorylated and lysine-acetylated peptides have more degrees of freedom and therefore there are more options to fit decoy peptides, this procedure results in taking a higher threshold for modified than unmodified peptides. Nevertheless, to reduce the false positive rate, the spectra of modified peptides with a low Mascot score were manually inspected and modified peptides with a Mascot score <10 were removed from the data set.
Automatic peptide quantification
The Mascot result files were then exported to Mascot peptide html file format without any further filtering and loaded into MSQuant version 2.0a81 (Mortensen et al, 2010
) together with the respective raw mass spectrometric data files. Peptide abundance ratios were determined automatically by MSQuant using the dimethyl (‘light' (12
), ‘medium' (12
) and ‘heavy' (13
)) lysine labels in the ‘Quantitation mode' without any additional peptide or protein filters. Phosphorylation sites were localized using MSQuant's PTM scoring. Selected peptide ratios for which contaminant peptide signal intensities and non-co-eluting peptide pairs were detected were re-calculated by manually adjusting their LC elution time window in MSQuant.
Automatic acetylated peptide identification and quantification
Acetylation as well as the dimethyl labels target lysine residues and protein N-termini. To identify acetylated peptides, both of these modifications have to be chosen as variable modification in MASCOT. However, in contrast to the ‘Quantitation mode,' the selection of the dimethyl labels as variable modifications includes experimentally impossible combination of these labels at the peptide N-terminus and at the lysine side chain (e.g., light label at the N-terminus and heavy label at the lysine side chain). Together with oxidized methionines, these modifications add up to nine variable modifications, which is the maximum number of variable modifications allowed in Mascot (light, intermediate and heavy dimethyl labels on lysines and peptide N-termini, acetylation of lysines and protein N-termini and oxidized methionines).
The use of a large number of variable modifications in database searches is known to decrease the number of identifications at a fixed FDR. Therefore, we employed a strict filtering of the Mascot search results for peptides with inconsistent labels (i.e., ‘light' dimethyl N-terminus and ‘intermediate' lysine) and unlabeled lysines/peptide N-termini as they should be incorrect. As additional control, we also estimated the accuracy of the search to be above 99% by counting the number of peptides with a C-terminally acetylated-lysine (either an in vitro
artifact or incorrect identification as trypsin is not expected to cleave C-terminal to acetylated-lysine residues (Choudhary et al, 2009
) in the entire acetylation data set. We only found seven such cases among the 759 non-redundant acetylated peptide matches (=0.9% FDR).
For automatic quantification of lysine-acetylated peptides in MSQuant, all ‘K' entries corresponding to acetylated-lysines in the Mascot result (.dat) files were replaced by ‘J' in the respective peptide sequences, and ‘Acetyl (J)' was added to the MSQuant parameter file (quantitationModes.xml).
Detection of outlier peptides
The detected unmodified peptides of a particular protein should all exhibit a similar abundance ratio. As the peptide ratios are used to determine the abundance change of the protein, the quality of the determined changes in protein abundance relies on reproducible quantification of the peptides that originate from the same parent protein. We called an unmodified peptide ‘outlier' in case it displays a significantly (P
-value<0.05, corrected for multiple testing according to Benjamini and Hochberg (1995)
) different ratio than the remaining peptides originating from the same parent protein. These different abundance changes can arise from co-eluting contaminants in the chromatogram for one of the isotope entities, stoichiometric dynamics in PTM or not considered differences between the combined proteomes (although processed in parallel).
To account for mixing error, the measure signal intensities of each peptide were normalized, such that the sum of signal intensities for each dimethyl label are equal in each proteome combination. Then, the change in protein abundance was estimated by the median of the peptides quantified for each protein and the normalized peptide signal intensities were corrected for this estimated protein abundance change of the parent protein. The corrected peptide signal intensities were analyzed with the R package OutlierD according to the author's recommendations (‘linear' method and k
=1.5 (Cho et al, 2008
)) and detected outliers were removed (Supplementary Figure S10
The change in protein abundance was calculated as the ratio of the sum of the peptide signal intensities normalized for mixing error. The change in protein abundance was calculated as the weighted average of the peptides identified for this protein. A minimum of two unmodified non-outlier peptides was required and outlier peptides as well as modified peptides were excluded from the protein quantification.
Detection of regulated peptides and regulated proteins
The normalized peptide signal intensities were corrected for protein abundance determined by weighted average. The R package OutlierD (Cho et al, 2008
) was used to determine the first and third quartiles of the peptide abundance changes, respectively. This information was used to calculate the z
-score and subsequently the P
-value for each peptide. The P
-values were then corrected for multiple testing according to Benjamini and Hochberg (1995)
. A modified peptide was called regulated if the corrected P
-value was <0.001 or a log2 abundance ratio was >1.5 for phosphorylated peptides and two for acetylated peptides. Essentially the same approach was used for the proteins. As only peptides were measured, but not entire proteins, the sum of peptide intensities was used as ‘protein signal intensity.' Proteins were considered ‘regulated' if the log2 abundance change was >1.5 and a corrected P
Integration of protein quantification from different proteome combinations
The peptide signal intensities from both technical duplicates were added for each protein, as they should represent maximal reproducibility. To integrate the biological duplicates the more significant change was taken. If the P-values were equal, the more severe change was chosen. In case neither of the two abundance changes was significant, the mean of the abundance changes from the biological duplicates was taken and the P-value was set to ‘none significant.' The regulated proteins were classified according to in which mutant the protein is regulated. Essentially the same method was applied to integrate the abundance change of modified peptides.
Functional enrichment analysis
The classification of the M. pneumoniae
proteins in the different clusters of orthologous groups of proteins was extracted from the ‘whog.txt' file downloaded from the National Center for Biotechnology Information (ftp://ftp.ncbi.nih.gov/pub/COG/COG/
) and the P
-value for the enrichment was determined using Fisher's exact test. The P
-values were corrected for multiple testing according to Benjamini and Hochberg (1995)
. Proteins were considered to be enriched in a particular cluster if the corrected P
-value was <0.05.
The molecular weight, the isoelectric point, the hydrophobicity and the instability index were calculated for each M. pneumoniae
protein annotated in SwissProt using the protparam tool from ExPASy (http://www.expasy.ch/tools/protparam.html
). The distribution of each parameter was then binned into 20 equally spaced bins. Supplementary Figure S3
shows the comparison of the coverage of each of these bins across all identified proteins in this study and all proteins annotated in the SwissProt database.
PTM scores from MSQuant were summed for each possible PTM site within phosphopeptides identified in multiple fractions. The PTM was localized if the highest PTM score was at least 1.25 times the second highest PTM score and the combined score was >4.
Evolutionary conservation of modified residues
PTM sites were localized in alignments of orthologous groups from eggNOG (Muller et al, 2010
) version 2. Species were assigned to one phylogenetic group being ‘other Mycoplasma
s,' ‘other Firmicutes,' ‘other Bacteria' or ‘Archaea and Eukaryotes' based on NCBI Taxonomy. If at least one protein form one species has the same amino acid as the PTM site, the site is considered ‘Site conserved' in this species, otherwise if in a window of three residues, one before to one after the PTM site, the same amino acid is found the site is considered ‘Conserved in window' in this species. Otherwise, the site is considered ‘Not conserved' in this species. If there is no protein for the considered species in the orthologous group, it is not counted at all. Each species is counted only once for one PTM site. The conservation level for each phylogenetic group is the number of ‘Site conserved' divided by the sum of ‘Site conserved,' ‘Conserved in window' and ‘Not conserved.' Random conservation was estimated by taking 10 times the same number of PTM sites with the same amino-acid distribution from the M. pneumoniae
proteome and performing the same conservation analysis.
Clustering of modified regulated peptides and proteins
Log2 ratios for regulated proteins and modified peptides in three k.o./wt comparisons and two k.o./k.o. comparisons were used to calculate uncentered correlations between each set of regulated proteins, regulated phosphopeptides and regulated lysine-acetylated peptides. The uncentered correlations were used for hierarchical clustering with hclust as implemented in the R package.
Network generation and analysis
Interactions between M. pneumoniae
proteins were derived from STRING (Jensen et al, 2009
) version 8.3. Scores between COGs and NOGs were converted to scores between M. pneumoniae
proteins by mapping M. pneumoniae
proteins to COGs and NOGs as they are in STRING v8.3. A network was generated between proteins regulated on abundance, phosphorylation and acetylation level by taking only interactions with a score >0.7. Random networks were generated by taking 100 times the same number of proteins from all M. pneumoniae
proteins and counting the number of interactions between them reveals a random expectation of number of interactions.
Minimum path lengths to HprK, PknB and PrpC were calculated for all M. pneumoniae proteins through the complete network of interactions (>0.7). Number of affected (upregulated or downregulated) proteins at each minimum path length were calculated for each k.o. strain and divided by the total number of proteins at each minimum path length.
Validation of lysine acetylation: TAP purification and quantitative western blot
One-liter cultures were incubated in five 300 cm2
cell culture flasks (Sarstedt) and harvested 96 h after inoculation. Cells were washed twice with ice-cold PBS and centrifuged at 9860 g
. Pellets were resuspended in 2 ml lysis buffer (50 mM Tris pH 7.5, 5% glycerol, 1.5 mM MgCl2
, 100 mM NaCl, 0.2% NP40, 1 mM DTT, 1 mM AEBSF, 1 mM PMSF, 1 μg/ml pepstatin A, 1 μg/ml antipain, 2 μg/ml aprotinin, 1 μg/ml leupeptin and 16 μg/ml benzamidin) and lysed mechanically using a douncer. TAP purification was done following established protocols (Kuhner et al, 2009
) stopped after TEV elusion. The elusion was split into two and each of these samples was analyzed by SDS–PAGE and western blot. A peroxidase anti-peroxidase antibody (Sigma, P1291) was used to detect the TAP-fusion proteins. Acetylated proteins were detected using an anti-acetyl-lysine primary antibody (Immunechem) and a HRP-coupled secondary antibodies (Sigma). For quantification, total band intensity was integrated with Photoshop software (Adobe) and normalized versus the highest detected peak (Supplementary Figure S2
Validation of protein abundance changes by quantitative western blot
strains were lysed as for the TAP purification. Total cell lysate of each of the four strains was then analyzed by SDS–PAGE and western blot using polyclonal antibodies raised against endogenous M. pneumoniae
proteins. Final detection was done using secondary antibodies coupled to HRP (Sigma). For the quantification, total band intensity was integrated with Photoshop software (Adobe) and normalized versus the highest detected peak (Supplementary Figure S5
Evaluating the protein complexes by separation on sucrose gradient, GF chromatography and western blot
Lysis was performed as mentioned for the TAP purification supplementing the lysis buffer with the deacetylase inhibitors nicotinamid (10 mM) and butyric acid (50 mM). Volume of 30 μl of samples were layered on a top of 4 ml sucrose gradient (10–35%) and separated by 14 h centrifugation at 130 000 g at 4°C. The gradient was subsequently divided into 22 fractions per 165 μl. GF chromatography was performed at 10°C on a Pharmacia SMART system at a flow rate of 40 μl/min by using a SuperoseTM 6 PC 3.2/30 column, equilibrated with lysis buffer. The chromatographic profile was monitored at 280 nm by using the μPeak monitor (Pharmacia). Volumes of 50 μl of samples were loaded on a column and 60 μl fractions were collected. Fractions from both sucrose gradient and GF were analyzed by SDS–PAGE and western blot (). Polyclonal antibodies produced in rabbits have been used to detect the GroS protein (Mpn574) and 50S ribosomal protein RlpA (Mpn220); final detection was done using secondary antibodies coupled to HRP (Sigma) on Image Station 4000 MM Pro (Kodak). For the quantification, the band intensities detected with Photoshop software (Adobe) were first normalized for equal sample loading and subsequently each fraction was represented as a percentage of total protein amounts of particular sample. The values obtained from three independent sucrose gradient separation experiments were used for calculation of average values and standard deviations. To test for a significant difference in the intensities for the three different groups (WT, PrpC, PknB), we performed a one-way ANOVA with a significance threshold of P<0.01. To further test for pairwise differences, we then applied Tukey's Honest significant difference test for the corresponding fractions, with a significance threshold of P<0.05.
Structure models of each sequence, where available, were retrieved from ModBase (Pieper et al, 2009
). ModBase gives several models for each UniProt entry, covering different parts of the sequence and with different scores, e
-values and sequence identity between the target and the template (template=the known structure on which the target is modeled). For each of the modified residues (Supplementary Tables S2 and S3
), and for their unmodified equivalents (unacetylated-lysine, unphosphorylated serine, threonine and tyrosine), the model with the lowest e
-value that included the residue was taken. For each of these models, NACCESS (http://www.bioinf.manchester.ac.uk/naccess/
) was used to calculate the relative accessible surface area (RSA) of all side chains, and a residue was defined as exposed when RSA>5%.
3D structures of interactions
Structural templates for interactions of any pair of proteins were found by BLAST comparisons (Altschul et al, 1990
) of the sequences against sequences of structures in the PDB (E
0.001), including biounit assemblies, and looking for pairs of proteins that hit different but interacting parts of the same structure. A particular pair of proteins might have zero, one or more templates. For each template, the residues of one component that are in contact with the other were found and, via the sequence alignments given by BLAST, used to infer the residues in contact in the query pair (with InterPreTS (Aloy and Russell, 2003
)). The occurrence of modified residues (Supplementary Tables S2 and S3
) in interfaces was compared with that of their unmodified equivalents and significance measured with a χ2
test. Images were produced with PyMol (http://sourceforge.net/projects/pymol/
). For selected protein interfaces, we also constructed homology models using MODELLER (Sali and Blundell, 1993
Microarray analysis of k.o. strains
A custom DNA array was used consisting of 688 70 mers representing 688 ORFs. The M. pneumoniae
array consists of oligos (70 bases, amino linker) spotted on the array four times each. The design process was done in cooperation with Operon Biotechnologies, synthesis of the 688 oligos (probes) was performed by Operon Biotechnologies and the spotting was done at the EMBL Genomics Core Facility (Guell et al, 2009
). M. pneumoniae
M129 was grown in 150 cm2
tissue culture flasks with 100 ml of modified Hayflick medium with the following composition: 18.4 g of PPLO broth, 29.8 g of HEPES, 10 g glucose, 5 ml of 0.5% phenol red and 35 ml of 2 N NaOH per liter. Horse serum and penicillin were included to a final concentration of 20% and 100 U/ml, respectively. In the reference condition, cells were grown for 96 h at 37°C.
After growth, surface-attached cells were washed once with PBS and immediately lysed in the cultivation flask by adding RLT buffer from the QiagenTMRNeasyPlus Mini Kit (Cat. Num. 74134). This isolation method used for RNA extraction removed most RNAs <200 nucleotides, thus preventing the synthesis of cDNA from tRNA. For cell lysis, 2 ml of RLT buffer in the presence of 0.134 M β-mercaptoethanol was used per cultivation flask. The purification was done according to the manufacturer's protocol.
In all, 9 μg of total RNA was used for the reverse transcription polymerase chain reaction (RT–PCR) using SuperScriptTM Indirect cDNA Labeling System from Invitrogen. This kit was used according to the manufacturer's indications, with the exception of two modifications. RT–PCR was carried out at 37°C instead of 46°C and the set of random hexamers (2 μl of 2.5 μg/μl) was used instead of polyT 20 mers. Hybridization and scanning was carried out at the EMBL Genomics Core Facility. Custom microarrays were scanned using an Axon GenePix 4000.