Synopsis of the systematic quantification of M. pneumoniae proteome. (A) Experimental design for the proteomic comparison of three M. pneumoniae deletion strains: hprK (red), pknB (yellow) and prpC (blue) with wild type (gray). The analyses account for biological variations and include several independent cultures of each strain (C1–C3). (B) Sample labeling and mixing scheme for the relative quantification of the differently perturbed proteomes. The various proteomes are differentially labeled with three stable isotopic forms of dimethyl, light (L), medium (M) and heavy (H). The mixing scheme includes both technical and biological duplicates. (C) Overall number of proteins and peptides identified and quantified. All PTMs data were normalized for changes in protein abundance (illustrated here for one protein). The fraction refers to (1) all ORFs annotated in Uniprot (UniProt Consortium, 2011), (2) the number of identified proteins, (3) the number of quantified proteins and (4) two peptides were identified with two phosphosites and two overlapping peptides cover one single phosphosite.

M. pneumoniae proteome, phosphoproteome and lysine acetylome. (A) Modified proteins are significantly enriched in functions related to metabolism and cellular processes and signaling. (B) Box plots indicating the relative extents of modification for serines, threonines, tyrosines and lysines within all observed modified proteins. (C) Modified proteins show a complex pattern of modification. Bubble plot showing the number of proteins with distinct modification profiles. (D) Phosphorylation is more positionally conserved in bacteria than lysine acetylation. The fraction of conserved modified residues is shown for either precise site conservation (site conservation) or a more plastic conservation within three residues (±1 amino acids conservation).

Impact of systematic kinase and phosphatase k.o. on M. pneumoniae phosphoproteome. (A) Regulated phosphosites are ordered according to whether they are (1) kinase regulated (PknB or HprK) (pink), (2) kinase and phosphatase regulated (blue) or (3) phosphatase regulated (orange). Heatmaps represent the log2 ratios of the phosphopeptides in the different strain, normalized for protein ratios. Phosphosites outside the box represent some kinase-regulated that phosphosites could not be measured in the phosphatase k.o.: they could be assigned to either class (1) or (2). Similarly, a few phosphatase-regulated phosphosites were not measured in both kinase k.o.. Strains and could belong to either class (2) or (3). (B) Representation of the three different phosphorylation steady states captured by the analysis. For the sites exclusively downregulated in the kinase k.o., the kinase activity largely overcomes that of the phosphatase, whereas for those also upregulated in the phosphatase k.o., kinases and phosphatase have balanced activity. Phosphosites only downregulated in the phosphatase k.o. are rare. They represent cases where the phosphatase activity largely overcomes that of the kinases. (C) Functional classification according to COG. The kinase-regulated substrates are enriched for metabolic functions, whereas the kinase and phosphatase-regulated substrates are enriched for Mycoplasma-specific functions.

Phosphorylation induces complex changes in protein abundance and patterns of lysine acetylation. The histogram represents the number of proteins or sites that show changing levels of phosphorylation (open blue bars), lysine acetylation (open purple bars) and protein abundance (open green bars) in the different deletion strains. The filled bars represent the fraction in phosphorylated proteins (blue fill), lysine-acetylated proteins (purple fill) or phosphorylated and lysine-acetylated proteins (yellow fill).

Multilevel impact of systematic kinases and phosphatase deletion on M. pneumoniae proteome. (A) STRING interactions (>0.7) of proteins regulated at the levels of protein abundance (green), phosphorylation (blue) and lysine acetylation (purple). Direct STRING interactions with HprK (red), PknB (orange) and PrpC (blue) are highlighted. These affected proteins are more frequently interacting with each other than expected from a random set of proteins. Bottom panels: examples of subnetworks. (B) Proteins were ordered by their shortest path to the k.o. proteins in the STRING network (>0.7). The average percentage, over the three k.o., of affected proteins decreases with increasing shortest path length.

PTMs are enriched at interaction interfaces, affecting protein–protein interactions. (A) Homodimerization of Mpn134 modeled on a homodimeric E. coli maltose transporter structure ((Oldham and Chen, 2011), PDB: 3PUY). Cyan and magenta ribbons show the interaction of two copies of Mpn134, spacefilled atoms colored by atom type show the side chains of S392 from both copies. Phosphorylation of S392 is predicted to lead to repulsion of negatively charged phosphates and likely prevent homodimerization. (B) Example of multiple modifications in a single interaction interface: EF-Tu (magenta) interacting with EF-Ts (cyan) modeled on E. coli EF-Tu–EF-Ts ((Kawashima et al, 1996), PDB: 1EFU). EF-Tu phosphorylation site T34 and EF-Ts acetylation site K133 are show as spacefilled atoms colored by atom type. Both are in the interface. (C) GoS/GroL chaperonin modeled on the structure from Thermus thermophilus ((Shimamura et al, 2004), PDB: 1WE3). Phosphorylation sites (T29) are in red spacefill and those of the acetylated-lysines in yellow spacefill. Those sites not in an interface are shown in orange (acetylation) or pink (GroL phosphorylation). One face has been removed to show the surface of the internal cavity. (D) A dimer of GroS heptamer modeled on the structure of a Chaperonin-10 tetradecamer from Mycobacterium tuberculosis ((Roberts et al, 2003), PDB: 1P3H). The overall structure is shown as a surface with one heptamer in blue to emphasize heptamer–heptamer interface. A single monomer is shown with magenta ribbons, with the C-α atom of the lone phosphorylated site (T29) in red spacefill and those of the acetylated-lysines in yellow spacefill. In this model, in at least one monomer, all these sites are in an interface (within 4 Å of a different monomer). Most occur on flexible loops whose conformations are expected to change between the template structure and different models. (C, D) Together show the possibility for some modifications to allow switching between different multimeric states and for those on the inner surface of the cavity to interact with substrate proteins. (E) Sedimentation of GroS on sucrose gradient. In the strain deficient in PrpC, the sedimentation profile of GroS (12 kDa) was found significantly affected; ^**P<0.01. This correlated with an increase in the level of T29 phosphorylation. For comparison, the sedimentation profile of RplA (Mpn220) remains largely unaffected (Supplementary Figure S9). The results of three independent experiments are represented.