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Molecular & Cellular Proteomics 7:299-307, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation^*,S

Boris Macek^,, Florian Gnad^,, Boumediene Soufi^¶, Chanchal Kumar, Jesper V. Olsen, Ivan Mijakovic^¶ and Matthias Mann^,||

From the Max Planck Institut for Biochemistry, Department of Proteomics and Signal Transduction, Am Klopferspitz 18, 82152 Martinsried, Germany; and ^¶ Technical University of Denmark, BioCentrum, Center for Microbial Biotechnology, DK-2800 Lyngby, Denmark

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.

Since the onset of molecular biology, Escherichia coli, a Gram-negative bacterium, has been one of the most used experimental cell systems; recently, it has become an invaluable host for heterologous gene cloning (8) and expression (9). Compared with other bacteria, studies of Ser/Thr/Tyr phosphorylation in E. coli commenced very early (7, 10). Two-dimensional gel experiments with protein extracts labeled with radioactive phosphorus suggested as many as 128 different Ser/Thr/Tyr phosphorylated E. coli protein spots. However most of them were only described in terms of pI and molecular weight (7) and, surprisingly, were never identified. Instead, other organisms, such as B. subtilis (5) and Corynebacterium glutamicum (6), became models for studying protein phosphorylation in bacteria; consequently, there is little current knowledge on Ser/Thr/Tyr phosphorylation in E. coli.

Homologs of eukaryotic Ser/Thr kinases have so far been found in many bacterial species (11), but not in E. coli. In addition to a preliminary description of a protein kinase C-like activity (12), this organism possesses two well characterized Ser/Thr kinases: the isocitrate dehydrogenase kinase/phosphorylase (13) and the recently described YihE kinase (14). With respect to tyrosine phosphorylation, E. coli possesses two BY-kinases (15), Wzc and Etk (16), which are capable of auto- and substrate phosphorylation. Several E. coli proteins were also reported to autophosphorylate on Ser/Thr residues, including the molecular chaperone DnaK (17), an essential GTPase termed Era (18), and the nucleotide diphosphate kinase (19). Presently, available data include 12 phosphorylation sites on 6 proteins in E. coli, obtained from individual biochemical studies (20).

Here we analyze the Ser/Thr/Tyr phosphoproteome of E. coli K12 using high accuracy MS in combination with phosphopeptide enrichment, and compare it with the phosphoproteome of B. subtilis. We report 81 phosphorylation sites on 79 E. coli proteins and show that the phosphoproteomes of these model bacteria have similar size and distribution of Ser/Thr/Tyr phosphorylation and share about 20% of phosphoproteins. We use these two datasets to study conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. Both phosphoproteomes show significantly higher conservation levels than a random protein population, with several phosphorylation sites being conserved from Archaea to humans, suggesting functional importance of this modification in prokaryotes as well as eukaryotes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Lysis—
Wild-type E. coli cells (strain: K12 isolate: MG1665) were grown in 4 L of Luria-Bertani (LB)¹ medium under vigorous shaking. At OD600 = 1, the cells were precipitated by centrifugation (5 min at 6000 x g) and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 5 mg/ml lysozyme, and 5 mM of each of the following phosphatase inhibitors: sodium fluoride, 2-glycerol phosphate, sodium vanadate, and sodium pyrophosphate (Sigma). Cell wall lysis was performed for 15 min at 37 °C, and cell membranes were disrupted by sonication. Dnase I (100 µg/ml) (Sigma) was added to the lysate and incubated for additional 10 min at 37 °C. N-octylglucoside detergent (Sigma) was added to final concentration of 1% for more efficient extraction and solubilization of membrane proteins. Cellular debris was removed by centrifugation at 25,000 x g for 30 min. The crude protein extract was extensively dialyzed against deionized water and lyophilized.

Protein Digestion and Phosphopeptide Enrichment—
The lyophilized crude protein extract (about 20 mg) was dissolved in denaturation solution (6 M urea, 2 M thiourea in 20 mM ammonium hydrogen carbonate), and prepared for phosphopeptide enrichment using strong cation exchange (SCX) chromatography, essentially as described previously (4). Briefly, proteins were reduced with dithiothreitol, alkylated with iodoacetamide, digested for 3 h with endoproteinase Lys-C (1/100 w/w) (Waco), diluted 4 times with 20 mM ammonium hydrogencarbonate, and further digested overnight with trypsin (1/100 w/w) (Promega). The resulting peptide mixture was divided in two parts that were analyzed separately as two technical replicates. The first stage of phosphopeptide enrichment was performed on a Resource S (GE Healthcare) SCX column as described previously (4). Ten SCX fractions were collected in each run and subjected separately to the second stage of phosphopeptide enrichment using TiO₂ beads (GL Sciences).

Before enrichment, SCX fractions and TiO₂ beads were pre-incubated with 2,5 dihydroxybenzoic acid (final concentration 5 g/L). About 10 mg of TiO₂ beads were added to each SCX fraction and incubated batch-wise for 60 min at room temperature. After washing twice with 80% acetonitrile/0.2% trifluoroacetic acid, peptides were eluted from the beads with 0.5% ammonium hydroxide solution in 40% acetonitrile (pH 10.5), almost completely dried in a vacuum-centrifuge and resuspended in 10 µl of 1% trifluoroacetic acid/2% acetonitrile in water for LC-MS analysis.

LC-MS/MS Analysis—
Liquid chromatography was performed on a 1100 nano-HPLC (Agilent) coupled to the LTQ-Orbitrap mass spectrometer (Thermo Fisher) as described previously (4). The LTQ-Orbitrap was operated in the positive ion mode, with the following acquisition cycle: a full scan recorded in the orbitrap analyzer at resolution R = 60000 was followed by MS/MS of the five most-intense peptide ions in the LTQ analyzer. Multistage activation was enabled in all MS/MS events to improve fragmentation spectra of phosphopeptides and "lock mass" option was enabled in all full scans to improve mass accuracy of precursor ions (21).

Data Processing and Validation—
Raw MS spectra were processed using Raw2msm software (21) and peak lists were searched against concatenated forward and reversed NCBI E. coli K12 protein database using Mascot search engine (Matrix Science). The following search criteria were used: full tryptic specificity was required; 2 missed cleavages were allowed; carbamidomethylation was set as fixed modification; oxidation (M), N-acetylation (protein), phosphorylation (STY), (H) and (D) were set as variable modifications; initial allowed precursor maximum mass deviation (MMU) was 10 ppm and fragment MMU was 0.5 Da.

All phosphopeptide spectra identified by Mascot were processed and validated using MSQuant software. Stringent acceptance criteria were used: besides good coverage of b- and y-ion series, only peptides of six or more amino acids were considered, and the maximum allowed mass deviation of precursor ion was 7 ppm. The probabilities for phosphorylation at each potential site were calculated from the posttranslational modification (PTM) scores, as described previously (4, 22). Phosphorylation sites that were occupied with probability > 75% are reported as identified ("Localization p value" = 1 in the supplemental Table I).

Enrichment Analysis of Gene Ontology Categories—
The E. coli identifiers were mapped to their UniProt counterparts using "EcoGene" database (http://ecogene.org). The Gene Ontology (GO) terms associated with these Uniprot identifiers were extracted from EBI GOA site, which were used to create reference GO ontology in Cytoscape (23) ontology format. The Cytoscape plugin BiNGO (24) was used to perform enrichment analysis for the GO categories. The analysis was done using "Hypergeometric test," and we selected all GO terms that were significant with p < 0.05 after correcting for multiple term testing by "Benjamini and Hochberg false discovery rate."

Evolutionary Analysis—
Homologous proteins to E. coli and B. subtilis proteins across 70 species from Shigella flexneri to human were determined via BLASTP (25). Homology search was performed on protein databases of 9 archaea, 53 bacteria, and 8 eukaryotes (for analyzed species, see supplemental Table IV). Databases were retrieved from Swissprot (http://expasy.org) in case of Archaea and Eubacteria, and SGD yeast database (26), FlyBase (27), and IPI (28) in case of eukaryotes. BLAST alignments were classified to be significant if the corresponding BLAST E values were lower than 1E-5. To distinguish between paralogs and orthologs, a two-directional BLASTP approach was used (29). Global alignments between homologous proteins were generated by the Needle software (30), based on the Needleman-Wunsch algorithm (30, 31).

Data Presentation in the PHOSIDA Database—
Identified phosphoproteins, phosphorylation sites, and homology data were uploaded to the in-house curated PHOSIDA database (www.phosida.com) (22). In the evolutionary section of PHOSIDA, phosphorylated proteins showing two-directional homology between species are marked in green, proteins with one-directional homology are marked in blue, and proteins that could not be aligned are marked in red. Integration of global alignments and their corresponding proportions of identities is also provided to enable users to define more conservative criteria for homology. In addition, PHOSIDA provides information on phosphorylation site conservation between species.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our laboratory has developed a high-accuracy, high-sensitivity method to study the phosphoproteome of any species. We have previously applied this approach, based on enrichment and direct analysis of phosphopeptides rather than whole phosphoproteins, to both eukaryotic (22, 32) and prokaryotic (4) Ser/Thr/Tyr protein phosphorylation. In this study, we used this generic peptide-centric approach for analysis of Ser/Thr/Tyr protein phosphorylation in E. coli at the phosphorylation site level (Fig. 1a).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Overview of the analytical workflow used in this study and a representative MS/MS spectrum. a, Overview of the analytical workflow used in this study. Trypsin digestion of the whole cell lysate was followed by enrichment of phosphopeptides using two stages of chromatography (SCX and TiO2). Phosphopeptides were separated on nano-HPLC and mass-measured and fragmented in the high performance LTQ-Orbitrap mass spectrometer. b, MS/MS spectrum of the phosphopeptide HRDLLGApTNPANALAGTLR from the nucleoside diphosphate kinase (ndk). Precursor (peptide) ion mass was measured in the orbitrap mass analyzer with mass deviation of 0.09 ppm. Rich backbone fragmentation in the MS/MS spectrum localized the phosphorylation site to Thr93.

Annotated spectra of all detected phosphopeptides are presented in the supplemental Fig. 1.

Phosphoproteome of E. coli—
Using this approach, we detected 105 phosphopeptides from 79 E. coli proteins with an estimated false positive rate of less than 1%. Furthermore, the precise site of phosphorylation in the fragmented phosphopeptide was determined with probability higher than 75% for 81 phosphorylation sites (Table I). Very high mass accuracy was crucial to reaching these values, and we achieved an average absolute mass accuracy of the phosphopeptides of 1.03 ppm, despite their low abundance. A representative MS/MS spectrum of a detected phosphopeptide is presented in Fig. 1b. Details about identified phosphopeptides are listed in supplemental Table I. In the identified phosphopeptides, a total of 55 serines, 19 threonines, and 7 tyrosines were found to be phosphorylated, yielding a Ser/Thr/Tyr phosphorylation ratio of 67.9%/23.5%/8.6%. Information on the classes of detected phosphoproteins was obtained using GO analysis and is summarized in supplemental Table II. Detected phosphoproteins were significantly over-represented in the main pathways of carbohydrate metabolism (p = 1.2 E-5), protein biosynthesis (p = 9.6 E-5), and phosphoenolpyruvate-dependent phosphotransferase system (PTS) (p = 1.3 E-4). Although proteins from all parts of bacterial cell were detected, membrane proteins were under-represented (p = 4.9 E-7). No special attempt was made to target this protein population, and this finding may simply reflect the commonly observed undersampling of membrane proteins because of their biophysical properties, rather than their lower phosphorylation levels.

To check whether detected phosphorylation sites in E. coli conform to known target sequences of eukaryotic protein kinases, we matched their surrounding motifs with 33 known target sequences from 25 eukaryotic protein kinases (supplemental Table III). As in the case of B. subtilis (4), we did not detect significant enrichment in any of the tested kinase target sequences, which points to different substrate specificity of bacterial and eukaryotic Ser/Thr/Tyr kinases.

Comparison of E. coli and B. subtilis Phosphoproteomes—
Having very similar genome sizes, E. coli and B. subtilis present good models for direct comparison of the general features of the Ser/Thr/Tyr phosphoproteome in taxonomically distant Eubacteria. Interestingly, their phosphoproteomes show striking similarity in size, number of detected phosphorylation events, distribution of Ser/Thr/Tyr phosphorylation, classes of phosphoproteins, and protein essentiality (Table II). Protein conservation analysis between these two phosphoproteomes revealed 14 direct orthologous proteins: elongation factor EF-G (fusA), phosphoglucosamine mutase (glmM), phosphoenolpyruvate-protein phosphotransferase of PTS (ptsI), protein chain elongation factor EF-Ts (tsf), nucleoside diphosphate kinase (ndk) (Fig. 1b), phosphopentomutase (deoB), malate dehydrogenase (mdh), pyruvate kinase I (pykF), glucosephosphate isomerase (pgi), enolase (eno), phosphoglycero mutase III (gpmM), phosphoglycerate kinase (pgk), D-tagatose 1,6-bisphosphate aldolase 2 (gatY), and phosphate acetyltransferase (pta). Most of these proteins are involved in glycolysis, showing that Ser/Thr/Tyr phosphorylation is particularly enriched in this metabolic pathway. Four orthologous phosphoproteins (glmM, ndk, pykF, and gpmM) contained conserved phosphorylation sites that were detected in both organisms. One-directional sequence alignments identified homologous phosphoproteins of an additional five E. coli phosphoproteins in B. subtilis: transaldolase B (talB), aspartate-semialdehyde dehydrogenase (asd), and fused fructose-specific PTS enzymes (fruB, ptsA, and dhaM).

This approach showed that the phosphoproteomes of E. coli and B. subtilis are highly conserved throughout all analyzed species. Details about the conservation of the E. coli phosphoproteome are summarized in the supplemental Table IVa. More than a half of E. coli phosphoproteins were found to be conserved in other bacterial organisms, in contrast to about 25% of the nonphosphorylated proteins (Fig. 2a). As expected, the highest conservation was found within -Proteobacteria and the highest degree of homology in the closely related Shigella flexneri, where 91% of the identified phosphoproteins had a homolog. Nearly 40% of E. coli phosphoproteins were also conserved in eukaryotes, in contrast to around 14% of the nonphosphorylated proteins. Unlike in prokaryotes, the proportion of conserved phosphoproteins was very constant throughout eukaryotic species. The extent of homology in Archaea was comparable with that of eukaryotes: 31% of E. coli phosphoproteins were conserved at the phosphoproteome, compared with only 13% at the proteome level.

View larger version (15K): [in this window] [in a new window]   FIG. 2. The evolutionary conservation of the phosphoproteomes. a, E. coli. b, B. subtilis. Protein databases of 9 archaeal, 53 bacterial, and 8 eukaryotic species were used to perform the evolutionary analysis. The conservation is reported as an average conservation of the phosphoproteome in all tested species from each domain.

Evolutionary Conservation of Bacterial Phosphorylation Sites—
The simplest and most common way to analyze conservation of protein sequences is direct application of the BLAST algorithm. However, BLAST presents a heuristic method to find significant local, rather than global, alignments. Because phosphorylation sites are mainly located in rapidly evolving loop regions, we first checked evolutionary conservation at the protein level. To identify orthologs and distinguish between orthologs and paralogs, we used a two-directional BLAST approach. To cover the entire protein sequence alignments of orthologs, we then applied Needle (30), a program based on the Needleman-Wunsch algorithm (31), to study the evolutionary conservation at the phosphorylation site level.

Phosphorylation sites in E. coli and B. subtilis were on average more conserved than their nonphosphorylated counterparts. This observation is most evident in Archaea: in the E. coli dataset, 44% of phosphorylated serines were conserved, in comparison to 23% of nonphosphorylated serines (Fig. 3a). Accordingly, 54% phosphothreonines were conserved in comparison to 28% of their nonphosphorylated counterparts (Fig. 3b). In the B. subtilis dataset, 32% of phosphorylated serines were conserved, in comparison to 24% of nonphosphorylated serines (Fig. 4a) and 62% of phosphothreonines were conserved in comparison to 29% of nonphosphorylated threonines (Fig. 4b). Because of their low number, we could not draw any statistically significant conclusions about phosphorylated tyrosines.

View larger version (18K): [in this window] [in a new window]   FIG. 3. The evolutionary conservation of the phosphorylation sites from the phosphoproteome of E. coli. a, conservation of phosphoserine. b, conservation of phosphothreonine.

View larger version (19K): [in this window] [in a new window]   FIG. 4. The evolutionary conservation of the phosphorylation sites from the phosphoproteome of B. subtilis. a, conservation of phosphoserine. b, conservation of phosphothreonine.

Interestingly, phosphorylated residues from the following nine proteins were found to be conserved from Archaea to humans: cysteinyl t-RNA synthetase (S270 in B. subtilis), phosphoglucomutase (S100 in B. subtilis; S146 in E. coli), nucleoside diphosphate kinase (T92 in B. subtilis; T93 in E. coli), pyruvate kinase (S36 in B. subtilis and E. coli), enolase (Y281 in B. subtilis), predicted GTP-binding protein (S16 in E. coli), D-3 phosphoglycerate dehydrogenase (T63 in E. coli), phosphoglucosamine mutase (S100 in B. subtilis; S102 in E. coli), and elongation factor Ef-Tu (T62 in E. coli). Note that observed conservation of these residues does not mean that they are actually phosphorylated in all species. However, because the roles of several of these phosphorylation events have been shown to be essential for function (34–36), it is likely that they are also present in many other organisms where the phosphorylation site is conserved.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Traditionally, PTMs on proteins are associated almost exclusively with eukaryotic rather than prokaryotic cells. However, a growing number of bacterial PTMs, partly uncovered through the increasing power of MS-based proteomics, lately received increased attention of the research community. Such modifications may be involved in hitherto unknown or underappreciated regulatory mechanisms of bacterial metabolic and signaling networks and perhaps present potential targets for new drugs. Here we have used powerful MS-based technology, previously used to detect several thousand phosphorylation events in mammalian cells (22) and about 100 phosphorylation sites in the Gram-positive bacterial model B. subtilis (4), to study the phosphoproteome of E. coli. By analyzing two distant bacterial phosphoproteomes, we reasoned that we could determine the generality of Ser/Thr/Tyr phosphorylation in bacterial and acquire sufficient data to study its conservation in taxonomically distant species.

Several insights obtained from the phosphoproteome of B. subtilis are now confirmed in the Gram-negative E. coli. We find that, in bacterial cells growing on LB medium, there are at least 80 proteins phosphorylated on Ser/Thr/Tyr residues. The exact number of phosphoproteins will vary between species and it is at present still unknown, but two-dimensional gel studies using radioactive detection and performed on the two microorganisms estimate it at not more than 150 (5, 7). In B. subtilis and E. coli, this presents about 5% of the annotated genes, which is a significantly lower number than in any eukaryotic organism, including yeast (32).

Most bacterial phosphorylation sites are on serine (70%) and threonine (20%), and a lesser number are on tyrosines (10%). Several proteins are multiply phosphorylated; however, this population is particularly scarce compared with eukaryotic cell, where multiple phosphorylated proteins are common and often serve as molecular "switchboards" for integration of signals. The virtual absence of these proteins in analyzed bacteria may not only reflect the much smaller number of phosphorylation events overall but also the fundamental difference in the role of Ser/Thr/Tyr in eukaryotic and prokaryotic cell: in the former it is primarily involved in signal transduction, whereas in the latter our data point to other cellular functions. Among bacterial phosphoproteins, there is a significant over-representation of enzymes involved in carbon metabolism (i.e. glycolysis, sugar transport) and essential proteins, which further supports the emerging view of Ser/Thr/Tyr phosphorylation as an important regulatory mechanism in the bacterial cell. One potential mechanism for such a regulation has already been described: phosphorylation of isocitrate dehydrogenase (icd) in E. coli occurs at Ser113, the residue involved in binding of the substrate (isocitrate), and therefore abolishes the enzyme action (37). Another such example may come directly from our study: the Thr93 residue of the essential protein nucleoside diphosphate kinase (ndk), found to be phosphorylated in both E. coli and B. subtilis and conserved from Archaea to humans, serves as an ATP-binding site. It is therefore reasonable to speculate that its phosphorylation will have a dramatic impact on the enzyme function.

Several important practical considerations are invoked by the results presented here. With more than 100 detected phosphopeptides in E. coli, it is somewhat surprising that only a few protein kinases capable of phosphorylating proteins at Ser/Thr/Tyr residues have been identified in this organism. This apparent lack of kinases lead to a widespread notion that this type of protein phosphorylation is practically absent in E. coli and bacteria in general. Therefore, it is widely assumed that a given eukaryotic protein, synthesized in E. coli, would purify in a homogenous nonphosphorylated form. However, with the potential of E. coli to phosphorylate proteins at Ser/Thr/Tyr residues demonstrated in this study, this assumption should be reexamined when considering E. coli and other bacteria as heterologous expression hosts.

The growing dataset of bacterial Ser/Thr/Tyr phosphorylation sites and the lack of similarity between target sequences of prokaryotic and eukaryotic protein kinases clearly indicates the existence of protein kinases specific for the bacterial kingdom. Currently available protein phosphorylation predictors are based on eukaryotic phosphorylation data (38); and although there are instances where their predictions were successful in bacteria (39), they remain of limited use for these systems. Therefore, the need for bacterial-specific phospho-predictors is evident and our data may provide a basis for training such predictors.

The high degree of conservation of proteins that are phosphorylated in E. coli or B. subtilis suggests their early presence in the course of evolution. In addition, it reflects the general constraints on these proteins, which are involved in major pathways such as carbon metabolism. However, a significant number of phosphorylation sites differ in the phosphoproteomes of the Gram-positive and Gram-negative models, and the extent of overall homology is not uniform because of the high diversity within bacteria. This indicates that protein phosphorylation kept on evolving as bacteria developed their particular lifestyles to conquer new niches. Traces of this evolutionary process can also be seen in the versatility of bacterial-specific kinase families, which evolved from nonkinase ancestors, such as the BY-kinases (15), the bifunctional Ser/Thr kinase/phosphorylases, and the tyrosine kinases with a guanidino-phosphotransferase domain (40). One important evolutionary factor that has also to be considered when examining the conservation of phosphoproteins is gene loss in parasites, which is mainly caused by the availability of the essential proteins from the host (41, 42). An interesting example is Buchnera aphidicola, an intracellular symbiont of the greenbug aphid (43). Although closely related to E. coli., only 13% of the entire E. coli proteome has an ortholog in this bacterium. However, 41.25% of the measured phosphoproteins in E. coli are present in B. aphidicola, clearly showing a higher conservation of the phosphoproteome compared with the entire proteome. The higher conservation of the proteins present in phosphoproteomes of E. coli and B. subtilis is evident throughout all selected species. The E. coli phosphoproteome appears to be even more conserved; however, this is related to the fact that Gram-negative bacteria outnumber Firmicutes in the set of selected representative bacteria. Therefore, for example, the phosphoproteins that are required for the spore cortex synthesis appear as poorly conserved in the analyzed bacterial set. In addition, the proportion of hypothetical or unknown proteins with low conservation is much higher in the B. subtilis set of phosphoproteins so the conservation of B. subtilis proteins in general appears lower in Archaea and eukaryotes in comparison to E. coli.

Nine specific phosphorylation sites from this study are conserved from Archaea to humans. They are in cysteinyl t-RNA synthetase, phosphoglucomutase, nucleoside diphosphate kinase, pyruvate kinase, enolase, predicted GTP-binding protein, D-3 phosphoglycerate dehydrogenase, phosphoglucosamine mutase, and elongation factor Ef-Tu. This unusually high conservation suggests that the phosphorylation of these sites may be also conserved in all life forms, although this has still to be proven. Nevertheless, the high conservation suggests that phosphorylation is in each case indispensable for enzyme activity, which is in turn essential for the cell. Good examples are phosphoglucomutases and phosphoglucosamine mutases, where phosphorylation of a key serine residue is a part of the reaction mechanism in which the phosphate is being transferred to the reaction product (34, 35). Another example is the phosphorylation of the elongation factor Ef-Tu, which occurs after its binding to the A-site of the ribosome (36); this phosphorylation triggers the release of Ef-Tu from the ribosome and therefore occurs in a cyclic fashion to optimize the translation rate. These highly conserved essential phosphorylation events can hardly be considered regulatory events in themselves, but they illustrate how the earliest life forms immediately used protein phosphorylation in the housekeeping of their cells.

	FOOTNOTES

 
Received, July 9, 2007, and in revised form, September 17, 2007.

Published, MCP Papers in Press, October 15, 2007, DOI 10.1074/mcp.M700311-MCP200

¹ The abbreviations used are: LB, Luria-Bertani; SCX, strong cation exchange; PTM, posttranslational modification; GO, Gene Ontology; PTS, phosphoenolpyruvate-dependent phosphotransferase system; LC-MS, liquid chromatography-mass spectrometry.

^* This study was supported by the Max Planck Society (to M. M.), the EU Framework 6 "Interaction proteome" grant (to M. M., B. M.), and the Danish Natural Science Research Council (to I. M.).

^S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

These authors have equally contributed to this work.

^|| To whom correspondence should be addressed. Tel.: 49-89-8578-2557; Fax: 49-89-8578-2219; E-mail: mmann{at}biochem.mpg.de

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