Figure 1.
Proteomics approach used for phosphotyrosine profiling of E. coli.
EHEC O157:H7 strain TUV93-0 and E. coli K12 strain MG1655 were grown to stationary phase in DMEM. Total protein was trypsin-digested, peptides purified using reversed-phased chromatography and subjected to immunoaffinity (IP)-based phosphotyrosine enrichment. Tyrosine phosphorylated peptides were identified by high resolution LC-MS/MS analysis using LTQ Orbitrap XL and Velos instruments.
Figure 2.
Definition of E. coli tyrosine phosphorylation site motifs.
(A–E) Probability logos of significantly enriched phosphotyrosine site motifs extracted from 512 unique pTyr sites by aligning peptide sequences comprising 12 residues surrounding the phosphorylated tyrosine residue using Motif-X (p value<0.001). Site motif consensus sequences with variable residues indicated as x and the number of unique sites comprising each motif are indicated. (F) Sequence logos of the general residue representation surrounding the phosphorylated tyrosine residue from the 512 unique sites with residues above the midline being overrepresented and those below underrepresented constructed using Phophosite logo generator.
Figure 3.
Phosphotyrosine proteins are involved in various cellular processes.
Pie diagram depicting the functional classification of tyrosine phosphorylated proteins identified in E. coli according to biological processes as shown in table S5. The cell processes category includes cell division and stress adaptation.
Figure 4.
Phosphotyrosine proteins are central in the metabolic network.
Protein-centric network representations of E. coli K12 (A) and EHEC O157:H7 (B) metabolism with pTyr (red nodes) and non-pTyr (yellow nodes) proteins indicated. Nodes represent proteins and edges represent compounds produced by one protein and consumed by other protein. Only names for the phosphotyrosine proteins are shown. Density plots showing the distribution of centrality closeness of pTyr (red) and non-pTyr (yellow) proteins in the metabolic network are shown. The higher the closeness centrality of a given protein (node), the shorter is its geodesic distances to other nodes in the graph.
Table 1.
Identified phosphotyrosine proteins expressed from pathogenicity islands in EHEC O157:H7 listed as type III system (T3SS) and non-T3SS proteins.
Figure 5.
Tyrosine phosphorylation of SspA Tyr92 affects virulence phenotypes of EHEC O157:H7.
(A) Location of Tyr92 in dimeric SspA (PDB 1YY7 [42]). The structure of dimeric SspA is shown as blue ribbon diagrams. The hydrophobic residues Tyr92 and His85 of the functionally important surface-exposed pocket are shown in green and orange, respectively. The hydroxyl group of Tyr92 that is subject to phosphorylation is shown in red. The SspA structure was visualized using PyMOL (Schrödinger LLC). (B) SspA Tyr92 positively affects expression and secretion of T3SS proteins. The abundance of LEE-encoded proteins in whole cell lysates (lanes 1–4) and their abundance in culture supernatants (lanes 5–8) from cultures of wild type EHEC O157:H7 and isogenic sspA mutants were determined by western analyses as described in Material and Methods. Strains tested included the sspA mutant containing the vector control pSec10*, the sspA mutant expressing wild type SspA from pSspA and the SspA Y92F mutant from pSspAY92F. EspA, EspB, Tir, SspA and GroEL were detected using polyclonal antisera against the respective proteins. GroEL served as an internal control for the total amount of protein in cell samples, and for the precipitation of proteins in culture supernatants to which 100 ng of GroEL were added. (C) SspA Tyr92 positively affects the A/E phenotype of EHEC O157:H7. A/E lesion formation was assessed using the FAS test as described in Material and Methods. HeLa cells were co-cultured for 5 h with wild type EHEC O157:H7, an sspA mutant and the sspA mutant harboring the vector pSec10*, pSspA (SspA) and pSspAY92F (SspAY92F). The actin cytoskeleton of HeLa cells was stained with FITC-phalloidin for visualization of the A/E lesions. Representative images of fluorescence stained actin of infected HeLa cells are shown. Arrows indicate examples of A/E lesions.
Figure 6.
BY kinases Etk and Wzc are dispensable for the A/E lesion phenotype of EHEC O157:H7.
HeLa cells were co-cultured with wild type EHEC O157:H7, etk, wzc and etk wzc double mutant derivatives for 5 h followed by actin staining of infected cells using FITC-phalloidin to visualize A/E lesion formation. Representative images of FITC-phalloidin stained actin of infected HeLa cells are shown with arrowheads indicating A/E lesions.