Phosphoproteome Analysis of Streptomyces Development Reveals

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Phosphoproteome Analysis of Streptomyces Development Reveals Extensive Protein Phosphorylation Accompanying Bacterial Differentiation Angel Manteca,*,†,‡ Juanying Ye,‡ Jesus Sanchez,† and Ole Nørregaard Jensen*,‡ †

Area de Microbiologia, Departamento de Biologia Funcional and IUBA, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain ‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

bS Supporting Information ABSTRACT: Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif “x(pT)xEx”. KEYWORDS: Streptomyces, phosphoproteomics, differentiation, sporulation, LC MS/MS

’ INTRODUCTION The Gram-positive soil bacterial genus Streptomyces is characterized by its complex morphological differentiation, which resembles that of filamentous fungi, and by the ability to produce a wide variety of secondary metabolites, including biologically active compounds.1 4 Because of its complex morphogenesis and industrial and medical importance, Streptomyces serves as a prokaryotic model for the study of multicellular differentiation. After Streptomyces spore germination, a fully compartmentalized mycelium (MI12h) initiates development until it eventually undergoes a highly ordered programmed cell death (PCD)5 (Figure 1). The MI differentiates to a second multinucleated mycelium (MII24h), which starts to express the chaplin and rodlin genes encoding the proteins constituting the rodlet layer that provides the surface hydrophobicity necessary for growth into the air (aerial mycelium; MII72h).6 At the end of the cycle, septation of the extremities of aerial hyphae and differentiation of the latter into spores is taking place (Figure 1). The MI fulfills the vegetative role in Streptomyces, and the late MII constitutes the reproductive phase destined to sporulate.7 In eukaryotes, reversible protein phosphorylation at serine, threonine, and tyrosine residues is a dynamic post-translational modification with stunning regulatory and signaling potential.8 In contrast, protein phosphorylation in prokaryotes is less well characterized, and its biological significance remains to be demonstrated. In prokaryotes, histidine/aspartate phosphorylations are r 2011 American Chemical Society

common in two-component systems.9,10 Recently, large-scale Ser/ Thr/Tyr phosphoproteome studies were reported for Escherichia coli,11 Streptococcus pneumoniae,12 Klebsiella pneumoniae,13 Lactococcus lactis,14 Campylobacter jejuni,15 Pseudomonas aeruginosa,16 Bacillus subtilis,17 Halobacterium salinarum,18 Mycoplasma pneumoniae,19 Corynebacterium glutamicum,20 Streptomyces coelicolor21 and Mycobacterium tuberculosis22 (Table 1). Using large amounts (milligrams) of protein obtained during vegetative growth, these studies established the existence of Ser/Thr/Tyr protein phosphorylation in bacteria (Table 1). However, the extent and the biological function of protein phosphorylation in bacteria are in most cases only poorly defined. The role of protein phosphorylation in the regulation of virulence in S. pneumonia, K. pneumonia, and P. aeruginosa12,13,16 was demonstrated by comparison of the phosphoproteome from pathogenic and nonpathogenic bacterial strains. In the case of M. tuberculosis, protein phosphorylation was demonstrated to be dependent on culture conditions.22 Bacteria with complex life cycles, such as actinobacteria, cyanobacteria and myxococcales, represent the evolutionary origin of several protein domains that are known to regulate eukaryotic signaling pathways, including eukaryotic-like protein kinases23 (Ser/ Thr/Tyr kinase protein domains; IKK, DAP, IRAK, etc.). The S. coelicolor genome contains 7825 ORFs, including 47 predicted eukaryotic-like protein kinases. This is twice the number of kinases Received: August 10, 2011 Published: October 17, 2011 5481

dx.doi.org/10.1021/pr200762y | J. Proteome Res. 2011, 10, 5481–5492

Journal of Proteome Research

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Figure 1. Streptomyces developmental cycle in solid cultures. (a) Cross section of a 36 h colony (0.5 cm in diameter) of S. coelicolor stained with SYTO 9 and propidium iodide and observed under the confocal microscope. (b) Cell-cycle features of Streptomyces development. MI12h, first compartmentalized mycelium; MII24h, presporulating second multinucleated mycelium without hydrophobic covers; MII72h sporulating second mycelium. PCD, programmed cell death. The classical nomenclature (substrate and aerial mycelium) and the hydrophobic layers (in gray) are also indicated.

predicted from genomes of other well characterized bacteria, including E. coli and B. subtilis (Table 1). In addition, Streptomyces harbors 49 eukaryotic type protein phosphatases.24 Interestingly, eukaryotic protein kinase inhibitors impaired aerial mycelium formation and secondary metabolism in Streptomyces,25,26 suggesting a role of protein phosphorylation in the regulation of these processes. In order to test this hypothesis, we performed a large-scale phosphoproteome analysis of Streptomyces solid cultures at three distinct stages of the differentiation process. We demonstrated that the main targets of Ser/Thr/Tyr phosphorylations were sporulation proteins, transcriptional regulators, protein kinases, and other regulatory proteins. Streptomycetes produce most of the biologically active compounds used in biomedicine,1 4 and they can be considered as the most important natural source for these kinds of compounds. Secondary metabolite production is strongly linked to hyphae differentitation.7,27 Results presented here constitute the first step of the necessary work to understand the role of protein phosphorylation in the differentiation process of Streptomyces. Considering the intricate links between morphological differentiation and antibiotic production, this knowledge is also expected to lead to the conception of novel strategies to discover novel bioactive secondary metabolites in natural Streptomyces strains.

’ EXPERIMENTAL PROCEDURES Bacterial Strains and Media

S. coelicolor M145 strain was used in this study. Solid cultures were performed on Petri dishes (8.5 cm) with 25 mL of solid GYM medium (glucose, yeast/malt extract)28 that were covered with cellophane disks, inoculated with 100 μL of a spore suspension (1 107 viable spores/mL), and incubated at 30 °C. Sampling and Fractioning of S. coelicolor Cells throughout the Differentiation Cycle

The mycelial lawns of S. coelicolor M145 grown on cellophane disks were scraped off at different time points (12, 24, and 72 h) using a plain spatula: the 12 h time point correspondeds to MI; 24 h to presporulating MII; and 72 h to sporulating MII (Figure 1). Samples were lysed by boiling in 2% SDS, 50 mM pH 7 Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 7 mM β-mercaptoethanol, 0.5 mM PMSF, and 1% phosphatase inhibitor mixtures 1 and 2 (P2850 and P5726, Sigma); sample viscosity was reduced by sonication (MSE soniprep 150, in four cycles of 10 s, on ice); the sample was cleaned by precipitation with acetone/ethanol (sample/EtOH/acetone 1:4:4 v/v/v overnight at 20 °C; washing with EtOH/acetone/H2O 2:2:1 v/v/v), resuspended in water, dialyzed against large volumes of water (1 h at 4 °C with four water changes), quantified by the Bradford method,29 lyophilized in aliquots of 50 μg, and stored at 80 °C. 5482

dx.doi.org/10.1021/pr200762y |J. Proteome Res. 2011, 10, 5481–5492


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Table 1. Comparison of S. coelicolor Phosphoproteome with Other Published Prokaryotic Phosphoproteomesa bacteria

ORFs Ser/Thr/Tyr kinases protein (mg)b phosphoproteins phosphopeptides phosphosites pS (%) pT (%) pY (%) reference

Gram + S. coelicolor

∼7825

47

S. coelicolor

∼7825

47

B. subtilis

∼5024

Gram

127

260

289c

46.8

48

50

40

44

46

34

52

14

21

19

10

78

103

78

69.2

20.5

10.3

17

C. glutamicumd ∼2996

16

e

56

ns

ns

ns

ns

ns

20

M. tuberculosis

∼3918

11

e

301

380

500

40

60

0

22

L. lactis

∼2777

5

20

63

102

79

46.5

50.6

2.7

14

S. pneumoniae

∼2069

4

1

84

102

163

47.2

43.8

9

12

E. coli P. aeruginosa

∼4300 ∼5568

19 17

20 1.2

79 39

105 57

81 61

67.9 52.8

23.5 36.1

8.6 11.1

11 16

K. pneumoniae

∼5524

M. pneumoniaed ∼688 archaea

5 2

0.3

30

5.2

this work

81

117

93

31.2

15.4

25.8

13

0.3

63

16

17

53

47

0

19

C. jejunid

∼1654

0

e

36

58

40

29

63

8

15

H. salinarum

∼2784

4

20

26

42

31

84

16

0

18

a

The amount of protein used for the phosphoproteomic experiments, the number of genome ORFs, amount of eukaryotic type Ser/Thr/Tyr kinases (Conserved Domain database entries cl09925, cd00180, smart00220, pfam10494), numbers of phosphoproteins/phosphopeptides/phosphosites detected, and percentages of Ser/Thr/Tyr phosphorylation are shown. b Total amount used in the all LTQ-Orbitrap or 2D gel runs. c 263 phosphorylation sites were unequivocally assigned by manual inspection (see Experimental Procedures). Twenty-six phosphorylations could not be assigned to a specific position (Supporting Information Table S1). d Bacteria whose phosphoproteome was analyzed by mean of 2D gels and/or radiolabeling and/or immunostaining with phosphoamino acid-specific antibodies instead of LC MS/MS. e Not reported.

Protein Digestion and Phosphopeptide Enrichment

Lyophilized proteins (50 μg) were dissolved in Milli-Q water (25 μL), S-alkylated with iodoacetamide, and digested with trypsin (Promega; 1 μg per 50 μg of protein) overnight at 37 °C. In order to prevent interference with CPP (see below), we did not use any buffer; pH was adjusted to 8 with a solution of 2 M NH3 3 H2O. Calcium Phosphate Precipitation

Phosphopeptides were pre-enriched using CPP as previously described.30 The volume of the peptide solution (50 μg) was adjusted to 100 μL, and the pH was adjusted to 10. Eight microliters of 0.5 M Na2HPO4 and 2 μL of 2 M NH3 3 H2O were added and mixed followed by the addition of 8 μL of 2 M CaCl2. The solution was vortexed and centrifuged at 20000g for 10 min at room temperature. The supernatant was removed, and 120 μL of 80 mM CaCl2 was added to suspend and wash the pellet. After a new centrifugation, the washing solution was removed, and the resulting pellet was dissolved in 40 μL of 5% formic acid, desalted using reverse phase chromatography (POROS R3 resin), and subjected to TiO2 enrichment. Phosphopeptide Enrichment with TiO2 Microcolumn

We follow the protocol described by Thingholm et al.31 Purified peptides were dried prior to LC MS analysis. LC MS/MS Analysis and Database Searches

LC MS/MS analysis was performed using a nanoliter flow EasyLC system (Thermo Fisher Scientific, Odense, Denmark) coupled with a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Peptides analyzed on an inhouse packed fused silica column (16 cm length, 100 μm inner diameter, 375 μm outer diameter, ReproSil, C18 AQ 3 μm; Dr. Maisch, Ammerbuch, Germany). The peptides were eluted with a 50 min gradient of 0 34% B solvent (A solvent: 0.1% formic acid, B solvent 0.1% FA in 90% acetonitrile). Peptide masses were measured in the Orbitrap at a resolution of 60 000 (m/z 400 2000). Up to five of the most intense peptides were selected from each MS scan and fragmented using multistage activation in the linear ion trap.

Raw data were processed using Proteome Discoverer software (version 1.1, Thermo Fisher Scientific). The resulting mgf files were searched against the NCBInr protein sequence database (December 02, 2009, 10487346 sequences) using S. coelicolor as the taxonomy (8573 sequences) through an inhouse Mascot server (version 2.2.06, Matrix Science, London, UK), using the following parameters: tryptic peptides with up to two missed cleavage sites were allowed; 5 ppm mass tolerances for MS and 0.5 Da for MS/MS fragment ions; carbamidomethylcysteine as fixed modification; and protein N-acetylation, oxidized methionine, and phospho_STY (serine, threonine, and tyrosine) permitted as variable modifications. Phosphopeptides were considered as potential candidates if they scored above the MASCOT homology threshold (average false positive rate for the 24 LC MS/MS runs of 1.3%; average significance threshold of 0.04). Candidate phosphopeptides were considered for further interpretation by manual inspection of their respective MS/MS spectra. Validation was carried out on the basis of the occurrence of at least four consecutive y or b ions and an intense signal should be assigned to ions produced by fragmentation at peptide bond N-terminal to proline if proline was present in the sequence. Phosphorylation sites were assigned by the appearance of a 69 Da/167 Da distance between fragment ions for phosphoserine and an 83 Da/181 Da distance for phosphothreonine. Phosphotyrosine-containing peptides were validated by the observation of a mass increase of 80 Da to unmodified peptide and the presence of the immonium ion at m/z 216 (singly charged) or a mass difference of 243 Da between fragment ions in spectra (Supporting Information Figure S2). Raw data were deposited in the EMBL-PRIDE database (accession numbers 16479 16502). Data Processing

Two biological replicates of each developmental stage (MI12h, MII24h, and MII72h) were processed (six samples in total). Phosphopeptides were enriched by using TiO2 and CPP/ TiO2 (methodological replicates), and each enriched sample was processed twice (technical replicates) in the LC MS/MS (24 LC MS/MS runs in total). When a phosphopeptide was 5483



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Figure 2. Overview of the analytical workflow used in this study, unique phosphopeptides, and representative MS and MS/MS spectrum. (a) Overview of the analytical workflow used in this study. Mycelium of three developmental time points were lysed by boiling in SDS and sonication; proteins were precipitated with acetone/ethanol; trypsin digestion was followed by phosphoenrichment using TiO2 and CPP/TiO2. Phosphopeptides were separated on nano-HPLC, mass-measured (MS), and fragmented (MS/MS) in the high performance LTQ-Orbitrap mass spectrometer. (b) Number of unique phosphopeptides detected with TiO2 and CPP/TiO2. (c) Phosphorylation site motif analysis; frequency logo generated using motif-x for Thr phosphopeptides. (d) MS/MS spectrum of the phosphopeptide AAAEDpTAGEGPAAGADEAR from the Thr-phosphorylated ATPase SCO5717. (e) Left, examples of the quantification strategy used in this work; abundance values (counts per second; average ( standard deviation) of MS precursor ions of representative phosphopeptides. Right, relative abundance of the nonphosphorylated proteins (iTRAQ ratios) estimated in a previous work.35 See text for details.

detected more than once, we retained the one having the highest MASCOT score. The ProteinCenter 2.0 software (Proxeon, Odense, Denmark) was used to conduct the computational and bioinformatic data analyses and protein classification. Proteins were classified manually into functional categories according to their annotated functions in the Gene Bank database and by homology/functions according to the Gene Ontology, the Conserved Domain, and the KEGG Pathway databases. When a protein was involved in the synthesis of any secondary metabolite, it was classified in the secondary metabolism group, even if it was included in additional categories. Functional partnerships between Streptomyces phosphoproteins were analyzed using the STRING database.32 The GPMAW software33 was used to determine the amino acid composition of the total number of identified phosphopeptides. Phosphorylation motifs were searched using the motif-x software.34 Kinase domains were identified using the Conserved Domain database; amino acid alignments (MUSCLE)

and maximum likelihood phylogenetic trees (PHYML) were made using the free online platform phylemon (http://phylemon. bioinfo.cipf.es/). Quantitative Analysis

The phosphopeptide abundances (counts per second) were analyzed only for the most reliable phosphopeptides: those detected and sequenced in the two biological replicates analyzed. Average abundances were estimated using the 24 LC MS/MS runs performed. In the runs in which the precursor ion was not sequenced, they were identified using the LC retention time points and the molecular masses from the runs in which they were sequenced; we used 0.14 min tolerance for LC retention time points and 5 ppm mass tolerance for precursor ion masses. Most phosphopeptides were detected in the CPP/TiO2 phosphoenrichments (see below and Figure 2b). In order to reduce the bias generated by the absence of phosphopeptides in the TiO2 phosphoenrichments, we only used the CPP/TiO2 samples to 5484



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Table 2. S. coelicolor Phosphoproteins Assigned to MI12h, MII24h, and MII72ha function stage

primary metabolism

MI12h

SCO4734

MII24h

SCO3403

secondary metabolism

transporters

SCO0498

SCO3115

stress proteins

regulatory proteins

septation, cell division

unknown

SCO4761

SCO3828

SCO4277

SCO5480

SCO6661

SCO5243

SCO1517

SCO5217

SCO1646

SCO5544

SCO5608

SCO5562 MII72h

SCO2198

SCO3850

SCO2368

SCO0588

SCO2950

SCO6096

SCO3671

SCO1630

SCO3103

SCO4296

SCO4117 SCO4969

SCO3288 SCO3289

SCO5249

SCO4242

SCO3542 SCO4721 SCO5357

SCO2077

SCO6162

SCO1860

SCO5199 SCO5834

MII24h

SCO4710

SCO7443

SCO5419

MII72h

SCO3067

SCO2079

SCO1192

SCO3859

SCO2082

SCO1749

SCO5717

SCO1919 SCO2168 SCO3302 SCO5507 SCO5864

MI12h

SCO1554

MII24h

SCO3037

MII72h

SCO4703

SCO2975 SCO4762

SCO4009 SCO6626

SCO3404

SCO5128 SCO5396

SCO7324 a

All these proteins harbored at least one phosphopeptide sequenced in two biological replicates and with an abundance in the developmental stage at which they were assigned at least five times greater than in the other stages (see Experimental Procedures and Supporting Information Table S1 for phosphopeptides and phosphorylation sites). Developmental stages (MI and MII), developmental time points, and protein functions (according to the Gene Ontology, Conserved Domain, and the KEGG Pathway databases) are indicated. See Supporting Information Table S1 for detailed description of the phosphorylation sites, phosphopeptides, and phosphoproteins.

estimate the average abundance and standard deviations. In the case of the few phosphopeptides identified only in the TiO2 samples, abundances were calculated using these samples. Abundance from each phosphopeptide was considered as significant only if their average ( standard deviation intervals were not overlapping among the MI12h, MII24h, and MII72h (Supporting Information Table S1). These phosphoproteins were assigned to a specific developmental stage when their abundances were at least five times greater than in the other stages (Table 2 and Supporting Information Table S1).

’ RESULTS AND DISCUSSION Conditions and Methodology Used for the Detection of S. coelicolor Phosphoproteome

The Streptomyces phosphoproteome was investigated at three distinct developmental stages: vegetative (MI12h), presporulation (MII24h), and sporulation (MII72h) (Figure 1).5 Two biological replicates of each mycelial stage were obtained according to our previous protocol35 (see Experimental Procedures). For each stage, phosphopeptides were enriched using either titanium dioxide (TiO2) affinity chromatography31 alone or TiO2 in combination with calcium phosphate precipitation (CPP).30 Each sample was analyzed twice (technical replicates) by LC MS/MS for a total of 24 LC MS/MS runs (Figure 2a). We identified a total of 259 phosphopeptides originating from

127 phosphoproteins at an estimated false discovery rate of 1.2% (MASCOT decoy database search option) (Tables 2 and 3). The phosphorylated amino acids (phosphorylation sites) were validated by manual inspection of phosphopeptide MS/MS data (see criteria in Experimental Procedures). A total of 263 distinct phosphorylation sites were identified, whereas 26 phosphorylation features could not be assigned unequivocally to a specific position (Supporting Information Table S1). Eighty percent of the detected phosphopeptides were recovered using the CPP/TiO2 protocol, demonstrating the utility of the CPP before TiO2 affinity chromatography, whereas 13% of the phosphopeptides were detected by both methods (Figure 2b). Our new experimental approach based on CPP and TiO2 proved highly efficient and sensitive for bacterial phosphoproteome analyses. Previous studies used milligrams of protein starting material, whereas we used only microgram levels. More importantly, we identified more phosphopeptides and annotated more phosphorylation sites than in other bacterial phosphoproteome studies, with the exception of recent studies of M. tuberculosis22,36 (Table 1). However, in the latter case,22 a total of 152 LC MS/MS analyses were performed to annotate 500 phosphorylation sites, whereas only 24 LC MS/MS runs were performed in the present study to identify 289 phosphorylation sites in S. coelicolor (Table 1). 5485



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Table 3. S. coelicolor Phosphoproteins Not Assigned to Any Developmental Stagea function primary metabolism

secondary metabolism

transporters

stress proteins

regulatory proteins

SCO1643

SCO1935

SCO2257

SCO0560

SCO1504

SCO0793

SCO1998

SCO4058

SCO0999

SCO2666

SCO0965

SCO2075

SCO5058

SCO1207

SCO3042

SCO1421

SCO2736

SCO6682

SCO1836

SCO3043

SCO1843

SCO3670

SCO5289

SCO2177

SCO3472

SCO5584

SCO2265

SCO3617

SCO5642

SCO2668

SCO3835 SCO4439

SCO6020 SCO6792

SCO2805 SCO3169

SCO4564

SCO7463

SCO3375

SCO4570

SCO7647

SCO3401

septation, cell division

unknown

SCO3822

SCO4730

SCO3862

SCO4808

SCO4088

SCO4965

SCO4281

SCO5369

SCO4330

SCO5535 SCO5591

SCO4509 SCO4967

SCO6754

SCO5173 SCO5389 SCO5464 SCO6745 SCO7132

a

Phosphoproteins detected only in one biological replicate or whose abundances were not significantly quantified (see Experimental Procedures) are shown. Protein functions (according to the Gene Ontology, Conserved Domain, and the KEGG Pathway databases) are indicated. See Supporting Information Table S1 for detailed description of the phosphorylation sites, phosphopeptides, and phosphoproteins.

Analysis of Phosphoproteome Data

Using the combination of CPP/TiO2 and high-performance mass spectrometry, we detected numerous phosphorylated proteins. The phosphopeptide abundances (counts per second) were analyzed only for the phosphopeptides that were detected and sequenced in both of the two biological replicates, and they were assigned to a specific developmental phase (MI12h, MII24h, and MII72h) if their abundance values were at least five times greater than in the other stages (see Experimental Procedures for details). Proteins harboring at least one phosphopeptide confidently quantified are those discussed below. We detected Thr phosphorylation of SCO5717 during the MII24h and MII72h phases (Figure 2d). The specific biological function of this phosphorylation event remains unknown, but mutation of the SCO5717 gene was recently reported to reduce cell growth and delay aerial mycelium formation (MII72h) in S. coelicolor.37 We also detected other proteins previously reported to be phosphorylated in Streptomyces or other bacteria: GroEL (SCO4762 and SCO4296) and DnaK (SCO3671), which were previously characterized in Streptomyces granaticolor;38,39 RsbR (SCO7324), an antisigma factor that was shown to be phosphorylated in B. subtilis;40 phosphorylation of FtsZ (SCO2082), which is essential for mycobacterial cell division;41 and DivIVA (SCO2077), which was shown to be phosphorylated in Mycobacterium42 (Table 2). These observations proved the efficiency and sensitivity of our phosphoproteomic approach. In order to investigate whether any particular protein functional classe was enriched in our phosphoproteome data set, phosphoproteins were grouped into functional categories

(see Experimental Procedures). Streptomyces phosphoproteome was enriched in regulatory proteins (transcriptional regulators, kinases etc.), whereas the proteome, analyzed without any phosphoenrichment prior to LC MS/MS (345 proteins described in ref 35), was mainly constituted of primary metabolism proteins (aerobic energy production, glycolysis and gluconeogenesis, pentose phosphate pathway, amino acid metabolism, etc.) (Figure 3). Phosphorylated proteins included well-characterized regulators of aerial mycelium differentiation and sporulation (Figure 4): Sig H (SCO5243), a protein regulating the onset of aerial mycelium differentiation;43 Ram S (SCO6682), the precursor protein of SapB, a well characterized protein involved in Streptomyces hydrophobic covers formation;44 FtsZ (SCO2082), DivIVA (SCO2077), and FtsH2 (SCO3404), proteins that are essential for hyphae septation during sporulation.41,45,46 This is the first time that these important regulatory proteins were described to be phosphorylated in Streptomyces. The analysis of the biological role of their phosphorylations will undoubtedly provide new insights about Streptomyces differentiation. Analysis of Phosphorylation Sites

Next, we analyzed the Streptomyces phosphorylation sites and putative phosphorylation motifs. The distribution of pS, pT, and pY was 47, 48, and 5%, respectively, which is in accordance with observations made in other bacteria (Table 1) and demonstrates a higher occurrence of pT and pY than in mammalian cells (86% pS, 12% pT, and 2% pY, respectively).47 Phosphorylation motifs (sequons) in Streptomyces were analyzed using the Motif-X server.46 5486



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involved in nucleoid organization and in pleiotropic regulation of gene expression.51 The fourth cluster encompasses the pT-protein SCO5717 (involved in cell growth and aerial mycelium formation),37 DiviVA (SCO2077), which is a pS protein regulating hypha septation (see below), as well as pT proteins of unknown function (SCO3115, SCO5173, SCO5128). Further experiments are necessary to unravel the biological function of the uncharacterized phosphoproteins present in these clusters, as well as the role played by the site-specific phosphorylation in the function of the proteins. Comparative Analysis of Bacterial Phosphoproteomes

Figure 3. Percentages of phosphoproteins (black bars) and nonphosphorylated proteins (gray bars) grouped into the gene ontology functional categories. Proteins were manually classified into functional categories according to their annotated functions in the Gene Bank database and by homology/function from the Gene Ontology, the Conserved Domain, and the KEGG Pathway databases. Primary metabolism (DNA/RNA replication, aerobic energy production, glycolysis and gluconeogenesis, pentose phosphate pathway, amino acid metabolism, nucleotide metabolism, translation, protein folding, RNA/protein processing, nucleases/RM methylases); secondary metabolism; transporters (ABC transporters, transporters and secreted proteins); stress and defense proteins; regulatory proteins (transcriptional regulators, kinases, other regulatory proteins); septation and cell division; unknown.

The motif “x(pT)xEx” was present in 18 phosphopeptides (Figure 2c) out of a total of 138 pT containing phosphopeptides (Supporting Information Table S1). This phosphorylation motif was not detected before in bacteria22 or eukaryotes,48 and to our knowledge, it may represent a new phosphorylation motif. We investigated the protein kinase profile of S. coelicolor by building a maximum likelihood (ML) phylogram including all the S. coelicolor eukaryotic-like protein kinases and key members of the eukaryotic kinase families49 (Figure 5). The majority of S. coelicolor protein kinases (28) grouped in three clusters of paralogues; the other nineteen were more diverse and highly divergent from eukaryotic kinases (Figure 5). Next, we used the STRING database32 to predict putative functional interactions between the newly identified Streptomyces phosphoproteins. Four different clusters of putative interacting proteins were retrieved (Supporting Information Figure S1). Two clusters consisted of well-characterized and functionally related proteins, namely chaperones/oxidative stress proteins and ribosomal proteins (Supporting Information Figure S1). The third and fourth cluster included transcriptional regulators and Thr phosphorylated proteins and were retrieved based on the co-occurrence criteria (protein interaction partners conserved across several organisms).32 The third cluster included putative transcriptional regulators (SCO3042, SCO3043, SCO6020) and several proteins of unknown function (SCO1421, SCO4088, SCO4330, SCO5199, SCO5864). It also included well-characterized proteins, such as RsrA (SCO5217), which is an antisigma factor involved in the regulation of mycothiol synthesis,50 and SCO3375, a homologue of the Mycobacterium Lsr2 protein

We compared the S. coelicolor phosphoproteome to the bacterial phosphoproteomes reported by other groups.11 22 Paralogues of 29 of the Streptomyces phosphoproteins (22.5% of the total) were detected in other bacteria (Figure 6). The Streptomyces phosphoproteome exhibited greater similarity with Gram-positive bacteria (21 proteins) (L. lactis, S. pneumoniae, B. subtilis, and M. tuberculosis), than with Gram-negative bacteria (8 proteins) (E. coli, K. pneumoniae, P. aeruginosa) or archaea (3 proteins) (H. salinarum) (Figure 6). Only 11 of the phosphoproteins detected by us in Streptomyces solid cultures were also found during the “mid-exponential growth” in nonsporulating liquid cultures (Figure 6).21 Despite the phosphoprotein differences that might reflect different growth conditions, other differences might be associated with bacterial differentiation. The “mid-exponential growth” corresponds to the vegetative stage (MI) from solid cultures,27 in which phosphorylation is low (Table 2). This suggests that the extent of Ser/Thr/ Tyr phosphorylation in bacteria might be underestimated because most of phosphoproteomic studies of bacteria were performed in the vegetative phases of these microorganisms.11 22 In consequence, phosphorylation events involved in or activated during bacterial differentiation have remained uncharacterized. Developmental Stage Specific Phosphoproteins

In order to compare the phosphoproteome from distinct Streptomyces developmental stages (MI and MII), we only considered the phosphoproteins harboring phosphopeptides sequenced in two technical replicates (63% of all) and with a good reproducibility of phosphopeptide ion abundances (see Experimental Procedures, Supporting Information Table S1). These phosphoproteins were assigned to the MI12h, MII24h, or MII72h if their estimated abundances were at least five times greater in one developmental stage than in the rest (Table 2 and Supporting Information Table S1). demonThe Streptomyces vegetative phase (MI12h) corresponds to the nondifferentiated mycelium, developing under optimal growth conditions (Figure 1),7 and is the phase with the lowest number of phosphorylation events (13 phosphoproteins, only 2 phosphoproteins detected exclusively in this phase) (Table 2, Supporting Information Figure S2). Streptomyces differentiation is triggered by stress conditions, and it is aimed at spore formation (Figure 1).5,52 Most of the protein phosphorylation events were detected in the differentiated hyphae (MII24h and MII72h) (64 phosphoproteins, 53 nondetected in the MI12h), which strates that phosphorylation plays an important and extensive role in Streptomyces differentiation (Table 2) (Figure 4, Supporting Information Figure S2). Key proteins regulating aerial mycelium differentiation (MII24h) and sporulation (MII72h) were differentially phosphorylated during development: transcriptional regulator SigH implicated in aerial mycelium differentiation (SCO5243) was phosphorylated at MII24h; FtsZ (SCO2082), which controls hypha septation and sporulation,41 was phosphorylated in MII72h; DivIVA 5487



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Figure 4. Integrated S. coelicolor phosphoproteome data mapped into a simplified metabolic network. Phosphoproteins shown are those belonging to well-characterized KEGG pathways (numbers in brackets), the Gene Ontology and Conserved Domain databases, or the literature. Phosphoproteins harboring at least one phosphopeptide assigned unequivocally to the MI12h (green), MII24h (yellow), and MII72h (red) (abundance values at least five times greater than in the other stages; phosphopeptides sequenced in both of the two biological replicates) are indicated (see Supporting Information Table S1 for specific phosphopeptides and phosphorylation sites). Phosphoproteins detected only in one replicate were not quantified and discussed in this work and are labeled with an asterisk. The rest of the phosphoproteins harbored phosphopeptides sequenced in two biological replicates, but their abundances were not significantly quantified according to the criteria described in Experimental Procedures.

(SCO2077), which is conserved in Gram-positive bacteria and is crucial for hyphae septation during vegetative growth and sporulation, 45 was phosphorylated at MII 72h ; and FtsH2 (SCO3404), a homologue of the E. coli FtsH protein involved in bacterial cell division,46 was phosphorylated during the MI12h

and MII24h/MII72h but in different residues in each developmental stage (Supporting Information Table S1). Transcriptional regulators, defense response proteins, receptors, and protein kinases were also phosphorylated during development and conceivably will be regulating Streptomyces differentiation (Table 2 and Supporting 5488



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Figure 5. Phylogenetic tree based on all the predicted S. coelicolor kinases (47) and key members of the eukaryotic kinase families (36) (S. cerevisiae). CAMK (DUN1 accession P39009, CMK1 accession EEU04203); AGC (RCK1 accession P38622, DBF2 accession P22204, KIN82 accession P25341, YPK1 accession CAA81967, TPK1 accession P06244); STE (MKK1 accession P32490, CDC15 accession P27636, STE20 accession CAY80273, SSK2 accession P53599, SSK22 accesssion P25390); CK1 (CDC7 accession NP_010267, CKA1 accession P15790); CMGC (SMK1 accession EEU04276, CDC28 accession NP_009718). Kinase domains were aligned using the MUSCLE software version 3.6, and the recovered ML phylogram is shown. The ML bootstrap values (%) for the main eukaryotic and Streptomyces kinase groups are shown. The scale bar indicates 0.1 substitutions per amino acid position.

Information Table S1). Several proteins involved in primary and secondary metabolism, as well as proteins of unknown function, were differentially phosphorylated during Streptomyces development (Table 2, Figure 4). SCO4734 and SCO4710 (ribosomal proteins) were phosphorylated at the phases in which they were less abundant (Figure 2e),35 which suggests that phosphorylation attenuates the remaining protein translational activity when ribosomal proteins are of low abundance in the cell. This is consistent with previous reports on the inhibition of translational activity by in vitro phosphorylation of Streptomyces ribosomal proteins.53,54

’ CONCLUSION We used, for the first time in bacterial phosphoproteomics, calcium phosphate precipitation (CPP) for phosphopeptide recovery prior to TiO2 and LC MS/MS analyses. CPP

proved to be extremely powerful for studying bacterial phosphorylation: using micrograms of protein (instead of milligrams as used in previous studies), we doubled and tripled the number of phosphoprotein identifications as compared to most of the published bacterial phosphoproteome studies. We demonstrated for the first time the active role of Ser/ Thr/Tyr protein phosphorylation in the differentiation of a bacteria species (Streptomyces). Most distinct phosphorylation events were detected during the presporulation and sporulation stages (80%); most of these phosphorylation sites in Streptomyces are reported here for the first time, including important regulatory proteins, and we identified a novel phosphorylation motif, x(pT)xEx. Our results constitute an important advance in understanding Streptomyces differentiation. Streptomycetes only produce 5489



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Figure 6. Overlap of S. coelicolor phosphoproteome from solid and liquid cultures, as well as the phosphoproteome of other well-characterized bacterial phosphoproteomes. Gram-positive bacteria (L. lactis, S. pneumonia, B. subtillis, M. tuberculosis); Gram-negative bacteria (E. coli, K. pneumonia, P. aeruginosa); and archaea (H. salinarum). S. coelicolor phosphoproteins with protein orthologous identified as phosphorylated in other bacteria are shown.

secondary metabolites when they are differentiated, and as a consequence, knowledge of the biochemical regulation of this differentiation process may contribute to the conception of novel strategies to discover new bioactive compounds that are greatly needed to meet the challenge of increased antibiotic resistance of pathogenic bacteria.

’ ASSOCIATED CONTENT

bS

Supporting Information Supporting Figure S1: Functional partnerships among Streptomyces phosphoproteins according to the STRING database. Supporting Figure S2: Number of phosphorylation sites, phosphopeptides and phosphoproteins assigned to the MI12h, MII24h, MII72h. Supporting Figure S3: Spectra of phosphopeptides. Supporting Table S1: Nonredundant phosphopeptides detected in S. coelicolor at different developmental stages (MI12h, MII24h and MII72h). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (A.M.); [email protected] (O.N.J.). Phone: (34) 985103000 ext. 5289. Fax: (34) 985103148.

’ ACKNOWLEDGMENT This research was funded by grant BIO2010-16303 from the DGI, Subdireccion General de Proyectos de Investigacion, MICINN, Spain. A.M. was supported by a postdoctoral grant from the Consejeria de Educacion y Ciencia (Asturias, Spain). Y.J. was supported by a grant from the Danish Research Agency (to O.N.J.). Research in the O.N.J. laboratory is supported by the Lundbeck Foundation and the Danish Research Agency.

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