Quantitative Proteome Analysis of Streptomyces coelicolor

Quantitative Proteome Analysis of Streptomyces coelicolor Nonsporulating Liquid Cultures Demonstrates a Complex Differentiation Process Comparable to That Occurring in Sporulating Solid Cultures Angel Manteca,*,†,‡ Hye R. Jung,† Veit Schwa¨mmle,† Ole N. Jensen,*,† and Jesus Sanchez‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Odense, Denmark, and Area de Microbiologia, Departamento de Biologia Funcional and IUBA, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain Received May 21, 2010

Streptomyces species produce many clinically important secondary metabolites and present a complex developmental cycle that includes programmed cell death (PCD) phenomena and sporulation. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that no differentiation took place. Recently, the existence of an early compartmentalized mycelium (MI) and a later multinucleated mycelium (MII) were described in solid and liquid cultures. The aim of this work was to compare the proteomes of the different developmental stages in liquid and solid S. coelicolor cultures, in order to give new insights in Streptomyces biology, and improve industrial fermentations. Using iTRAQ labeling and LC-MS/MS analysis of peptides, we demonstrate that differentiation in S. coelicolor liquid cultures is comparable to solid cultures. Eighty-three percent of all the identified proteins showed similar abundance values in MI and MII from liquid and solid cultures. Proteins involved in secondary metabolism (actinorhodin and type II polyketide biosynthesis, β-lactamases, epimerases) were up-regulated in MII. Proteins involved in primary metabolism (ribosome, Krebs cycle, and energy production) were detected in greater abundance in MI. The most remarkable protein abundance differences between MII from solid and liquid cultures were associated with the final stages of hyphae compartmentalization and spore formation. Keywords: proteome • iTRAQ • differentiation • Streptomyces coelicolor

Introduction Approximately two-thirds of industrial antibiotics and large numbers of eukaryotic cell differentiation inducers and inhibitors are synthesized by members of the Streptomyces genus.1–4 Streptomycetes undergo a complex developmental cycle, which includes sporulation in solid cultures; however, most Streptomycetes do not sporulate in liquid cultures. Industrial processes for secondary metabolite production are performed in liquid cultures (large bioreactors), and it is generally assumed that differentiation processes are absent under these conditions.5–9 The classical Streptomyces developmental model for confluent solid cultures assumes that differentiation processes take place along the transversal axis of the cultures (bottom-up): Completely viable vegetative mycelia (substrate) grow on the * Correspondence: Dr. Angel Manteca, Area de Microbiologia, Departamento de Biologia Funcional and IUBA, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. E-mail: [email protected]; phone, (34) 985103000, ext 5289; fax, (34) 985103148. Prof. Ole Nørregaard Jensen. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Odense, Denmark. E-mail, [email protected]; phone, (45) 65502368; fax, (45) 65502467. † University of Southern Denmark. ‡ Universidad de Oviedo. 10.1021/pr100513p

 2010 American Chemical Society

surface and inside agar until they undergo a programmed cell death process (PCD), after which hyphae differentiate to a reproductive (aerial) mycelium characterized by the presence of hydrophobic covers, giving it a characteristic grayish appearance.10 Substrate and aerial mycelia are multinucleated, but at the end of the cycle, aerial hyphae form septa and spore chains (Figure 1) (reviewed in Fla¨rdh and Buttner10). Recently, we have refined this developmental cycle describing novel aspects during the presporulation phases in liquid and solid cultures.8,11–17 We have characterized the existence of a previously unidentified compartmentalized mycelium (MI) that initiates the developmental cycle after spore germination.11,12 The MI suffers a highly ordered PCD, and the remaining viable segments of this mycelium begin to enlarge as a multinucleated mycelium (MII). In solid cultures, two types of MII have been defined, based on the absence (in early development) or presence (in late development) of the hydrophobic layers characteristic of aerial hyphae.18,19 The traditionally denominated substrate mycelium corresponds to MII lacking hydrophobic layers, and the aerial mycelium to MII coated with these hydrophobic layers (Figure 1).15 The only mycelial phases Journal of Proteome Research 2010, 9, 4801–4811 4801 Published on Web 07/19/2010

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Figure 1. Streptomyces coelicolor development stages and sample preparation. (a) Cell-cycle features of Streptomyces development. Mycelial structures (MI, first compartmentalized mycelium; MII, second multinucleated mycelium), liquid and solid cultures, the classical nomenclature of substrate and aerial mycelium, and the hydrophobic layers are indicated. (b) Confocal laser-scanning fluorescence microscopy pictures of the different types of mycelia: left, MI stained with the membrane stain FM4-64; middle, MIIL stained with the cell wall stain WGA; right, sporulating hyphae of MIIS stained with WGA. Developmental time points are indicated. Arrows indicate septa. See text for details.

present in liquid cultures were MI and MII without hydrophobic layers8,9 (Figure 1). Streptomyces biology has been studied using proteomics approaches in various cellular contexts, including PCD,14 germination,20,21 variations in the proteome of the bald A mutant,22–24 primary response to phosphate limitation,25 diauxic lag phase,26 and Streptomyces griseus A-factor mutant.27,28 Recently, we reported the first quantitative mass spectrometrydriven proteome analysis of Streptomyces differentiation in solid cultures,17 demonstrating the switch from primary to secondary metabolism in MI and MII. System biology experiments (transcriptomics, proteomics, etc.) in liquid cultures are imperative in order to understand Streptomyces differentiation in these conditions, and eventually to improve industrial fermentations. Recently, two independent transcriptomic analyses of Streptomyces gene expression levels as a function of time in liquid cultures were performed.29,30 These studies demonstrated the existence of a transition from primary to secondary metabolism during development in these conditions. In the present work, we go one step further by designing a protocol to fractionate the MI and MII developmental stages in liquid cultures, comparing the differences in their proteomes and also with the proteomes of analogous structures from solid cultures. This first comparative study of proteomes from Streptomyces hyphae in solid and liquid cultures revealed similarities and distinct differences between the MI and MII.

Experimental Procedures Bacterial Strains and Media. S. coelicolor M145 strain was used in this study. Liquid cultures were maintained in R5A liquid media.8 Flasks of 100 mL with 20 mL of culture medium were inoculated directly with spores (1 × 107 spores/mL) and incubated at 200 rpm and 30 °C. For solid cultures, Petri dishes 4802

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(8.5 cm) with 25 mL of solid GYM medium (glucose, yeast/ malt extract)31 were covered with cellophane disks, inoculated with 100 µL of a spore suspension (1 × 107 viable spores/mL), and incubated at 30 °C. This medium promotes the rapid development of a lawn that differentiates readily and yields abundant sporulation. Sampling and Fractioning of S. coelicolor Cells throughout the Differentiation Cycle. Cells from S. coelicolor grown in liquid cultures were processed at different developmental time points (14 and 90 h). The 14-h time point corresponds to the first compartmentalized mycelium (MIL) and 90 h to the second multinucleated mycelium (MIIL). Two independent cultures were prepared and processed (biological replicates). Samples (100 mL from 14 h culture and 20 mL from 90 h culture) were centrifuged at 5000g (10 min at 4 °C). Mycelial pellets were mechanically disaggregated (vigorous vortexing for 1 min) in 40 mL of A buffer (50 mM Tris-HCl, pH 7, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 7 mM β-mercaptoethanol, and 0.5 mM PMSF), precooled to 0 °C, and centrifuged for 10 min at 7740g and at 4 °C, 5000g. Usually, two washing steps are enough to separate the extracellular protein in Streptomyces,13 but we repeated the mechanical disaggregation and washing steps 8 times, in order to improve reproducibility. In the case of solid cultures, the mycelium lawn of S. coelicolor M145 grown on cellophane disks was scraped off at different time points (12 and 72 h) using a plain spatula. The 12-h time point corresponds to MIS and 72 h, to MIIS (Figure 1). As in liquid cultures, two independent cultures were prepared. At 12 h, the first compartmentalized mycelium was separated from the nonseptated mycelium by conversion of the cell compartments to protoplast forms.17 Samples of MII were obtained during phases in which MI had died (72 h). Mycelial pellets were mechanically disaggregated (vigorous vortexing for 1 min) in A buffer (2.5 g of mycelium in 10 mL) precooled to 0 °C, and

Streptomyces Proteome Variations during Differentiation

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washed as previously described. In all cases, samples were observed under confocal laser-scanning fluorescent microscopy after staining with vital dyes, as detailed below. Cells were broken up in an MSE soniprep 150, in 4 cycles of 10 s, on ice. The unbroken cells and cellular debris were eliminated by centrifugation (7740g) at 4 °C for 15 min. Cytosolic and membrane fractions were obtained according to Quiros et al.32 by ultracentrifugation at 100 000g in a Beckman LB-70 M ultracentrifuge. The resulting supernatant constitutes the cytosolic fraction, whereas the sediment represents the membrane fraction, which included membraneanchored proteins (intrinsic and extrinsic), as well as peripheral proteins weakly bound to the membranes. Membranes were resuspended in buffer A and incubated at 0 °C for 30 min with periodic vortex shaking. They were subsequently ultracentrifuged again at 100 000g. This process was repeated 3 times, discarding the supernatants. Membranes were later resuspended in 100 mM Na2CO3 (pH 11) and washed two more times. These three supernatants were collected and corresponded to the extrinsic membrane proteins. Finally, the membranes were washed two times with buffer A without salt. These membranous pellets corresponded to the intrinsic membrane proteins, which were not delipidated. Supernatants derived from the washing steps in Na2CO3 (extrinsic membrane proteins) were collected and dialyzed (Sigma D7884 benzoylated cellulose tubing) against buffer A at 4 °C for 1 h with four buffer changes. Membrane fractions were stored at -80 °C. Viability, Membrane and Cell Wall Staining. The permeability assay previously described for Streptomyces was used to stain the samples.33 This involves staining the dead cells with propidium iodide and viable cells with SYTO 9 green fluorescent nucleic acid stain (LIVE/DEAD Bac-Light Bacterial Viability Kit, Molecular Probes, L-13152). Equal volumes of the mixture of the stains and the mycelial sample were incubated in the dark for at least 10 min. The samples were observed under a Leica TCS-SP2-AOBS confocal laser-scanning microscope at a wavelength of 488 and 568 nm excitation and 530 nm (green) or 640 nm (red) emissions. Images were mixed using the Leica Confocal Software. Membranes were stained with the Lipophilic styryl dye, N-(3triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64) (Invitrogen, T-3166). Streptomyces cells were collected at various time points. Harvested mycelium was processed as described elsewhere.11 Cells were fixed in 2.8% paraformaldehyde and 0.0045% glutaraldehyde in PBS (0.14 M NaCl, 2.6 mM KCl, 1.8 mM KH2PO4 and 10 mM Na2HPO4) for 15 min at room temperature, and washed twice with PBS. FM 4-64 was added to a final concentration of 1 mg mL-1 and incubated at room temperature for 3 h. Samples were washed twice and observed under the confocal laser-scanning microscope at wavelengths of 550 nm excitation and 700 nm emission. Cell walls were stained with wheat germ agglutinin (WGA) conjugated with Texas red (Invitrogen W-21405), which binds selectively to N-acetylglucosamine and N-acetylneuraminic acid. Fixed cells were incubated for 1 min in 2 mg mL-1 lysozyme in glucose/Tris/EDTA (GTE: 50 mM glucose, 20 mM Tris/HCl, pH 8, 10 mM EDTA). The samples were washed again with PBS and blocked in 2% BSA in PBS for 5 min. WGA was added to a concentration of 100 mg mL-1 in 2% BSA in PBS, and incubated at room temperature for 3 h. Finally, the samples were washed eight times with PBS and observed under the

Figure 2. Overview of the mass spectrometry based proteomics strategy. The iTRAQ-reagent methodology used for multiplexed comparative analysis of Streptomyces proteins isolated by SDSPAGE during development. The six steps performed in parallel for the six gel slices are indicated by arrows. Developmental time points and subcellular fractions are indicated.

confocal microscope at wavelengths of 595 nm excitation and 615 nm emission. Protein Separation and iTRAQ Labeling of Peptides. Protein quantification was performed using the Bradford34 and Lowry35 assays with a bovine serum albumin standard (Sigma). Proteins were separated by SDS-PAGE (50 µg per lane) using precast PAGEr 4-20% Tris-Glycine gels (Lonza) and stained with Coomassie Blue (Coomassie Brilliant Blue G-250). For intrinsic membrane proteins, membranes containing 50 µg of protein were boiled in the SDS loading buffer for 5 min and loaded directly in the gel. The four samples (MIS, MIIS, MIL, and MIIL) of each subcellular fraction and the two biological replicates analyzed were loaded in six different gels, which were used for six independent iTRAQ experiments. Each gel-lane was divided into six slices with a scalpel. Gel slices were cut into small pieces, washed with distilled water, and shrunk with acetonitrile. Cys residues were reduced with DTT and S-alkylated with iodoacetamide, swelled with a solution of 10 ng/µL trypsin (Promega), 50 mM triethylammonium bicarbonate (TEAB) digestion buffer, and incubated overnight at 37 °C. After digestion, supernatants were recovered and peptide extractions from remaining gel fragments were performed with a volume of 5% formic acid for 30 min; subsequently, an equal volume of pure acetonitrile was added and incubated for an additional 30 min at room temperature. Extracts obtained from each gel slice were vacuum-dried. For quantification, peptides were labeled with iTRAQ 8plex reagent (Applied Biosystems, Foster City, CA) as per our previously reported protocol:17,36 113-, 114-, and 115-iTRAQ tags for 12-, 24-, and 72-h time points from solid samples, respectively, and 116 and 117 for liquid cultures, at 14 and 90 h of culture (Figure 2). After labeling for 2 h at room temperature (RT), samples were combined (6 samples corresponding to the original gel pieces). The concentration of organic solvent was reduced using a vacuum concentrator, and peptide desalting was performed using GELoader micropiJournal of Proteome Research • Vol. 9, No. 9, 2010 4803

research articles pet tips (Eppendorf) prepared with C18 (Empore extraction disks, 3M) and R3 (Poros Oligo R3). Analysis of iTRAQ Labeled Peptides by NanoLC-Tandem Mass Spectrometry. Tryptic peptides were separated using a NanoAcquity UPLC system (Waters Corporation) modified with a 2.6 µL PEEKSIL/sample loop (SGE, Darmstadt, Germany). Mobile phase A was 0.1% formic acid in ddH2O and mobile phase B was 0.1% formic acid in 90% acetonitrile (Fisher Scientific). A 2.5 µL sample was injected and loaded into the BEH C18, 1.7 µm, 15 cm × 75 µm analytical reversed phase column (Waters Corporation) in a direct injection mode with 3% B for 10 min at 400 nL/min. Peptides were eluted from the column with a linear gradient of 3-7% B for 4 min, 7-30% B for 60 min, 30-60% B for 15 min, 60-90% for 5 min, at a flow rate of 300 nL/min. The column was washed with 90% B for 10 min followed by equilibration for 14 min at a flow rate of 400 nL/min. The column temperature was maintained at 36 °C. The lock mass solution for MS and MS/MS was composed of 500 fmol/µL of (Glu1)-fibrinopeptide B (Sigma) and delivered by the auxiliary pump of the nanoAcquity equipment to the reference sprayer of the NanoLockSpray source of the mass spectrometer at a constant flow rate of 500 nL/min. The UPLC system was interfaced to a Q-TOF tandem mass spectrometer (Synapt, Waters Corporation, Manchester, U.K.). The mass spectrometer was operated in positive ion mode at a mass resolution of approximately 10 000 full width at halfmaximum (fwhm). The TOF analyzer (v-mode) of the mass spectrometer was externally calibrated with (Glu1)-fibrinopeptide B fragment ions from m/z 50 to 1500. Acquired data were postcalibrated using the doubly protonated precursor ion of (Glu1)-fibrinopeptide B. The reference sprayer was sampled every 120 s. LC-MS/MS data were acquired using a datadependent acquisition method. MS survey analysis was performed for 0.48 s with an interscan delay of 0.02 s followed by two MS/MS cycles. The fragment ions from the two most abundant multiply charged precursor ions (+2, +3, and +4) were detected at an integration rate of 0.48 s with a 0.02 s interscan delay. The collision energy ramped from 20 to 45 eV. Dynamic exclusion of precursors was set to 60 s. Each sample was analyzed twice. The precursor ions selected for the fragmentation during the first LC-MS/MS analysis were excluded in the second LC-MS/MS analysis. LC-MS/MS Data Analysis. The ProteinLynx Global server (PLGS) program version 2.3 was used to convert LC-MS/MS raw data into pkl files. Pkl files were submitted for search by the MASCOT search engine (version 2.2) against the NCBInr database with taxonomy limited to S. coelicolor (22 Jan. 2009, 8537 entries). The following MASCOT search parameters were used: peptide mass tolerance 10 ppm, fragment mass tolerance 0.1 Da, trypsin cleavage with a maximum of 2 missed cleavages. Fixed modification: S-carbamidomethyl on cysteine; iTRAQ on lysine residues and N-termini of peptides. Variable modifications: oxidation on methionine. The annotated mass spectrum of individual MS protein identification was shown in the Supporting Information. Peptide false positive rates were calculated using the decoy option provided by MASCOT (using the combined pkl file). Relative quantification was performed using PLGS (Waters Corp.) with automatic normalization. The PLGS quantification algorithm uses Bayesian Markov Chain Monte Carlo methods to explore the subsequent probability, and takes the different scores of individual peptides from a protein into account in order to quantify changes in expression. Results obtained from 4804


Manteca et al. PLGS were exported into MS-Excel for further computational and bioinformatic data analysis. Proteins that were not represented by any peptide above the MASCOT homology threshold were discarded. When a protein was detected more than once in the same biological replicate (six gel slices), we retained the one having the highest MASCOT score. For proteins identified in the two biological replicates analyzed, iTRAQ ratios were considered as significant if their average in both replicates (( standard deviation) was greater or lower than one. With respect to the remaining proteins, iTRAQ ratio values were considered significant if their coefficient of variation (CV) was less than 0.25. Consequently, we retained protein abundance values with good reproducibility between biological replicates (CV < 0.25), as well as those with CVs exceeding 0.25, but with averaged iTRAQ ratios that varied significantly between mycelial stages (average iTRAQ ratios ( SD above or below one); we discarded the remaining proteins (protein abundance values without good reproducibility between biological replicates). The ProteinCenter 2.0 software (Proxeon, Odense, Denmark) was used to conduct the computational and bioinformatic data analyses and protein classification. Proteins were classified 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. Cluster Analysis of Protein Expression Profiles. The averaged iTRAQ values obtained in two biological replicates for each protein at all four phases analyzed (MIS, MIIS, MIL, MIIL) were log2-transformed. Data were normalized to obtain a mean value of zero and a standard deviation of one, ensuring that proteins with similar expression patterns can be easily compared without taking into account their absolute values. For clustering, we used the fuzzy c-means algorithm with a Euclidean distance matrix.37 This method groups the data into c protein clusters with most similar patterns by minimizing an objective function. The results provide c membership values for each protein. A membership value gives a measure in the range (0, 1) of how closely a protein’s expression pattern follows that of the cluster center. We have considered clusters with membership values greater than 0.5 as significant. We associated each protein to the cluster for which it had the highest membership value. The parameter for the fuzzyness of the data was set to m ) 2. The optimum value for the other parameter, the number of clusters c, was determined by comparing the Xie-Beni index38 and the minimum centroid distance39 calculated from the corresponding results.

Results and Discussion Identification and Quantification of Streptomyces Proteins. Streptomyces is a mycelial bacterium with a complex developmental cycle which makes it difficult to fractionate different mycelial phases. We recently developed and reported a methodology to overcome this impediment in solid cultures.17 In the present study, we applied this methodology to obtain MI and MII phases from solid cultures (MIS, MIIS; 12 and 72 h, respectively). MI in liquid cultures (MIL) was obtained at early time points (14 h); MII in liquid cultures (MIIL) was obtained during phases after the death of MIL (90 h).8 In both cases, mechanical disaggregation of mycelial pellets combined with intensive washing removed the protein released by secretion or lysis. Samples were further fractionated into three subcellular fractions: cytosolic, membrane-anchored proteins (intrinsic


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Figure 3. Presentations of the quantitative proteomics data set obtained for developmental stages of S. coelicolor. Number of identified proteins, subcellular location, and quantitative proteomic data analysis are shown. (a) Venn diagram showing overlap of proteins identified in each of the two biological experiments. Number of proteins significantly quantified according to criteria described in Experimental Procedures is indicated. (b) Venn diagram of protein distribution in the subcellular fractions. These proteins correspond to those detected in two biological replicates. (c) Correlation of the values for biological replicates significantly quantified (see Experimental Procedures for details). Cytosolic, membrane, and extrinsic proteins were combined. (d) Variation of iTRAQ ratios (average from two biological replicates) of different developmental phases (blue, red and green), with respect to methodological variation (orange); proteins significantly quantified in all the conditions analyzed were shown (105 proteins); iTRAQ ratio values for each protein were sorted in increasing order. Cytosolic, membrane, and extrinsic proteins were pooled.

membrane proteins), and peripheral proteins bound to the membrane by weak bonds (extrinsic membrane proteins).17

from liquid or solid cultures (MIIL/MIIS, red line; or MIL/MIS, blue line) (Figure 3d).

Proteins were further processed for quantitative proteomics using isobaric tags (iTRAQ) and LC-MS/MS as previously reported (Figure 2).17 A total of 642 proteins (8.3% of S. coelicolor proteome) were identified from peptide MS/MS spectra that scored above the peptide MASCOT homology threshold value (false positive rates of 1.36% and 1.08% for each biological replicate) (Figure 3). In two biological replicates, 361 proteins were detected, and 359 were quantified in at least one of the developmental phases analyzed (MIS, MIIS, MIL, MIIL) (Figure 3a). These 359 proteins were distributed among the different subcellular fractions analyzed (Figure 3b) and were the only ones considered for further analyses and presented in the Tables and Figures included in this manuscript. The foldchange of all the quantified proteins were consistent across the biological replicates analyzed (Figure 3c). The analysis of the individual protein abundance values (average log10 iTRAQ ratios from two biological replicates) between different developmental stages (MIL/MIS, MIIL/MIIS, MIIL/MIL) (blue, red, and green lines in Figure 3d) demonstrates that they were far more variable than those acquired from a methodological replicate of the same developmental stage (MIL/MIL, orange line in Figure 3d). Moreover, disparities in the iTRAQ ratios between MII and MI in liquid conditions (MIIL/MIL; green line) (Figure 3d) were greater than those between the same mycelial stage obtained

Clustering of Proteins with Similar Abundance Profiles. With the aim of identifying the proteins with similar abundances along the four developmental phases analyzed, we used the fuzzy c-means algorithm33 with a Euclidean distance matrix (Figure 4). We clustered proteins that were detected in two biological replicates and only in one subcellular fraction (cytosolic or membranes) (223 proteins) (Figure 3). One hundred and forty-seven proteins were assigned to 6 specific clusters, each of which exhibited distinct protein profiles (Figure 4). Most of the proteins involved in primary metabolism were included in clusters 1 (13 proteins up-regulated in MIL and MIS) and 2 (26 proteins up-regulated in MIS): ribosomal proteins (SCO4711, SCO4719, SCO3909, SCO4718) (MII/MI ratios between 0.2 and 0.5), proteins of the Krebs cycle and energy production (SCO4475, SCO3092) (MII/MI ratios between 0.4 and 0.7), or proteins involved in lipid metabolism (SCO1815) (MII/MI ratio of 0.6) (Table 1). By contrast, proteins involved in secondary metabolism, as well as regulatory proteins, were included in clusters 3 and 4 (17 proteins up-regulated during the MII phases) and cluster 5 (4 proteins up-regulated during MIIL): proteins involved in actinorhodin biosynthesis (ActVA and ActVA4) (MII/MI ratios between 2 and 8), proteins implicated in the biosynthesis of type II polyketides (SCO5086) (MII/ MI ratios of 28 and 6), an epimerase (SCO0395) (MII/MI ratios Journal of Proteome Research • Vol. 9, No. 9, 2010 4805

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Figure 4. Cluster analysis (classification by fuzzy c-means approach) of protein expression patterns for S. coelicolor developmental stages. Clusters include proteins detected in only one subcellular fraction (pooled cytosolic and membrane peptides) with similar expression patterns along developmental time points. Number of proteins for each functional category, are indicated. Primary metabolism (DNA/RNA replication, aerobic and anaerobic energy production, glycolysis and glyconeogenesis, pentose phosphate pathway, amino acid metabolism, nucleotide metabolism, translation, protein folding, RNA/protein processing, nucleases/RM methylases, lipid metabolism); secondary metabolism (secondary metabolite synthesis and secreted proteins, DNA competence, TTA BldA targets, bld whi proteins); transporters (ABC and others); proteins with unknown functions; stress and defense proteins; regulatory proteins (transcriptional regulators, kinases and phosphatases, other regulatory proteins); catabolism/proteases; cell wall/membrane/septation (cell division and septation proteins; proteins involved in cell wall and membrane synthesis). Proteins clustered were those significantly quantified at least in one of the developmental phases analyzed (see Experimental Procedures).

of 1.9 and 2.2), a β-lactamase (SCO2380) (MII/MI ratios of 1.6 and 2.5), a histidine kinase (SCO4677) (MII/MI ratio of 3), and so forth. Proteins included in cluster 6 (Figure 4) displayed the maximum normalized protein abundance values in MI or MII, depending on the developmental conditions (liquid or solid cultures), and could not be unambiguously assigned to either MI or MII. These clusters were used to organize the proteins in Supporting Information Tables 1-3, and the same methodology was used to identify proteins with similar or different abundance patterns in the cytosolic or membrane fractions (Supporting Information Table 4). Overall, protein functions associated with primary and secondary metabolism clustered together, and their abundance values correlated well with Streptomyces differentiation. Proteins with similar functions are clustering together, and consequently, this knowledge will facilitate the interpretation of further experiments designed to define the biological role of proteins with putative or unknown function. Similarities and Differences between MI and MII Proteomes from Liquid and Solid Cultures. Next, we compared the MI and MII proteomes from solid and liquid cultures by considering the abundance values of the main functional protein groups (Figure 5). With few exceptions, the differentially expressed proteins showed similar relative abundances in solid and liquid conditions, that is, the same 4806


proteins were up- or down-regulated in MIIS and MIIL with respect to MI (83%) (Figure 5). The proteins with greatest divergence in their iTRAQ ratios between MI and MII in solid and liquid cultures (MII/MI log iTRAQ ratios greater than 0.2 or less than -0.2) are shown in Figure 6. Twenty of these proteins had similar abundance values in liquid and solid cultures (-0.2 < log iTRAQ MIL/MIS and MIIL/MIIS < 0.2) (Figure 6a, right panel), and can be considered as reliable markers of MI and MII (Figure 6a). The proteins detected in greater abundance in MI were involved in the Krebs cycle and energy production (SCO4475, SCO3092) (MII/MI ratios between 0.4 and 0.7); lipid metabolism (SCO1815) (MII/MI ratio of 0.6) (Table 1); a putative transport associated protein (SCO1903); aldehyde dehydrogenase (SCO4913); a putative TetR transcriptional regulator (SCO1691), and hypothetical proteins (SCO5414, SCO4179, SCO4033, SCO2067) (Table 1). SCO1691 is a very intriguing transcriptional regulator, which was only detected in MI (Table 1). Several TetR family transcriptional regulators have been described in Streptomyces as repressors of antibiotic biosynthesis and export,40–42 so a putative role of SCO1691 repressing the onset of antibiotic production in the MI might be feasible. Several proteins were more abundant in MII. We detected an epimerase (SCO0395) (MII/MI ratio of 2); proteins involved in lipid metabolism (SCO5385) (MII/MI ratios of 5 and 3);

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Table 1. Proteins Showing the Greatest Abundance Differences between MI and MII from Liquid and Solid Cultures (Figure 6)a MIIS/MIS

function

SCO no.

Krebs cycle and energy metabolism Amino acid metabolism Lipid metabolism

SCO3092 SCO4475 SCO2770 SCO1815 SCO5385 Other anabolic enzymes SCO6551 Translation, protein folding SCO4711 SCO4719 SCO3909 SCO4718 SCO4721 Unknown SCO4033 SCO2067 SCO4179 SCO4913 SCO5414 Stress/defense proteins SCO1903 SCO1925 SCO2035 ABC transporters SCO2008 Transporters/secreted Secondary metabolites synthesis

Transcriptional regulators

Cell division/septation Kinases Other regulatory proteins Bld Whi proteins

Cl

function

1 1 3 1 3 3 2 2 2 2 2 1 1 1 1 1 1 3 3 3

Oxidoreductase Cytochrome C assembly protein speB, agmatinase 3-oxacyl-(ACP) reductase 3-hydroxybutyryl-CoA dehydrogenase Aldo/keto reductase family 30S ribosomal protein S17 30S ribosomal protein S5 50S ribosomal protein L9 50S ribosomal protein L18 50S ribosomal protein L15 Hypothetical protein Hypothetical protein Hypothetical protein aldehyde dehydrogenase Hypothetical protein Possible transport associated protein Component of SufBCD complex Protein Disulfide Oxidoreductases ABC-type branched-chain amino acid transport systems ftsY, prokaryotic docking protein Epimerase/dehydratase Ketoacyl reductase; Biosynthesis of type II polyketide backbone ActVA4; Biosynthesis of actinorhodin Beta-lactamase ActVA; Biosynthesis of actinorhodin TetR transcriptional regulator CAP family transcription factor DeoR transcriptional regulator RNA polymerase sigma factor Possible secreted with DivIVA domain Spo0M homologue proteins Histidine kinase-like ATPases ssDNA-binding protein Whib family sigma factor

SCO5580 3 SCO0395 3 SCO5086 5 SCO5079 SCO2380 SCO5077 SCO1691 SCO0168 SCO4920 SCO4895 SCO5142 SCO1793 SCO4677 SCO3907 SCO5046

3 3 3 ns 6 3 ns ns 4 3 ns ns

C

I

MIIL/MIL

E

C

0.4 0.6

I

0.2

0.2 0.6 0.5

2.2

0.9 1.2

2.3

1.2 1 1.1

2.2 4.3

2.1

1.2

1.7

0.7 0.9 MISb 0.6 1.4

1.7 0.8

ns

1 1.5

ns 2.1 2

1.1 ns

0.7

3.1

2

1 2.2 0.8 2.3

2.3 2.3

1.5 2

1.5

1.6 3.8 MILb 0.4 2.1

1 0.9

1.2 1.3

2.6

2.5 8.4 MISb 1.5

0.8 1 0.8 1.2

2 3

1.9 1.9

0.9 0.8

0.8 1.2

2.2 3

0.4 0.4

1.1 0.8 0.7

E

0.8 0.8

0.8

0.6 2

I 1 1.4

1

0.4

0.6 2.1

C

0.9 1 0.8 1 0.9 0.8 0.7 0.7 1.3

0.7

0.6 0.6

MIIL/MIIS

E

0.9 0.9 1.4 0.8 0.4 0.7 0.7 0.5 0.5

0.4 0.5

I 0.7 1

2.7 0.6 3 1.9 0.6 0.6 0.5 0.5 1.1

2.8

C

0.7 0.7

3 0.6 5 1.6 0.2 0.4 0.4 0.4 0.4

0.4 2

MIL/MIS

E

ns 3.5 0.7

a See Supporting Information Tables 1-3 for details. The second multinucleated mycelial stages with respect to the first compartmentalized mycelium (MIIS/MIS, MIIL/MIL) are shown. iTRAQ ratios are the average of two biological replicates. MIL/MIS and MIIL/MIIS ratios are also indicated. C, cytosolic; I, membrane intrinsic; E, membrane extrinsic. n.s., nonsignificant iTRAQ ratio values or clustering (see Experimental Procedures). Functions (according to Gene bank, Gene Ontology, Conserved Domain, and KEGG); Cl, clusters of proteins with similar abundances (see Figure 5); C, cytosolic; I, membrane intrinsic; E, membrane extrinsic. b Proteins detected exclusively in MI.

disulfide oxidoreductases (SCO2035) (MII/MI ratio of 2); a component of the SulfBCD complex, which is believed to participate in iron-sulfur cluster formation during oxidative stress (SCO1925) (MII/MI ratio of 2);43 transporters (SCO5580, SCO2008) (MII/MI ratios between 2 and 6); SCO2770, speB, agmatinase (MII/MI iTRAQ ratio of 3), and SCO6551, an aldo/ keto reductase (MII/MI iTRAQ ratios of 1.6 and 1.9) (Table 1 and Figure 6a). Several regulatory proteins were also detected in greater relative abundance in MIIL and MIIS: SCO4920, a DeoR family transcriptional regulator (MII/MI ratio of 2); SCO5046, a whiB family sigma factor regulating the final sporulation steps (MII/MI ratio of 1.92) (Table 1). The greater abundance of SCO5046 in MII is consistent with its role in hydrophobic cover formation and sporulation in solid cultures, in addition to differentiation in liquid conditions.10 Only 5 of the proteins with the greatest differences in MI/MII iTRAQ ratios exhibited inverse relative abundances in liquid and solid cultures (Figure 6b). SCO1793 (sporulation Spo0M homologous protein) and SCO5142, homologue to the Bacillus sporulation protein DivIVA,44 were more abundant in MIIS than in MIIL (2.5-fold) (Table 1). It suggests that the most remarkable differences between MIIS and MIIL emerge in the final stages of hyphae compartmentalization and spore formation. The 50S ribosomal proteinL15(SCO4721),aputativessDNA-bindingprotein(SCO3907), and a putative transcriptional regulator (SCO0168) also showed different abundances in MI and MII obtained in liquid and solid

culture conditions (Figure 6b and Table 1). The biological functions of these proteins have yet to be determined. Relative Abundance of Proteins Detected in More than One Subcellular Fraction. Sixteen of the proteins detected in more than one subcellular fraction (cytosolic, intrinsic or extrinsic membrane) presented great divergences in their relative abundances between MI and MII (MII/MI log iTRAQ ratios greater than 0.2 or less than -0.2). Among these, only 6 proteins showed no variation between liquid or solid cultures (MIL/MIS and MIIL/MIIS log iTRAQ ratios between -0.2 and 0.2) (Figure 7), which can be added to the 20 proteins described above (Figure 6). Five of these proteins were detected in greater abundance in MII: a possibly secreted protein (SCO5995) with a TTA leucine codon (MII/MI ratios between 1.9 and 4), which could be one of the targets of the bldA developmental gene (the only S. coelicolor gene encoding tRNA for the TTA leucine codon);45 a putative transcriptional regulator (SCO3571) (MII/ MI ratios of 2); a DNA gyrase (SCO3873) (MII/MI ratios between 1.9 and 4); a putative secreted protein (SCO2780) (MII/MI ratios of 6), and an ABC transporter (SCO7677) (MII/MI ratios between 1.6 and 1.9) (Table 2 and Figure 7). In contrast, 30S ribosomal protein S1 (SCO1998) was detected in greater abundance in MI (MII/MI ratios between 0.2 and 0.6) (Table 2). SCO5995 and SCO7677 had signal peptides; SCO1998 and SCO2780 had transmembrane domains; however, SCO3571, and SCO3873 did not harbor any transmembrane domains or Journal of Proteome Research • Vol. 9, No. 9, 2010 4807

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Manteca et al.

Figure 5. Protein abundance values (logarithm of iTRAQ ratio values) for the main functional groups of proteins. Primary metabolism (DNA/RNA replication, aerobic and anaerobic energy production, glycolysis and glyconeogenesis, pentose phosphate pathway, amino acid metabolism, nucleotide metabolism, translation, protein folding, RNA/protein processing, nucleases/RM methylases); secondary metabolism (secondary metabolites synthesis, TTA BldA targets, bld whi proteins); transporters (ABC transporters, transporters and secreted proteins); stress and defense proteins; regulatory proteins (transcriptional regulators, kinases, other regulatory proteins). Proteins presented here are those detected in only one subcellular fraction (cytosolic or membrane) and were quantified in all developmental phases (see Experimental Procedures). MI, first compartmentalized mycelium; MII, second multinucleated mycelium; S, solid cultures, L, liquid cultures. Proteins labeled with asterisks are those included in Figure 6.

signal peptide. Further work will be necessary to define the biological significance of the presence of these proteins in more than one subcellular fraction. Several regulatory proteins were also identified among the proteins detected in more than one subcellular fraction (Figure 3b and Supporting Information Table 4): two components of the bldk ABC transporter complex (bldkB and bldkD) were down-regulated in MII with respect to MI (0.7-fold) (Table 2). The bald (bld) genes control the onset of aerial hyphae formation by regulating the expression of genes involved in the production of SapB,46–48 rodlins,18 and chaplins.19 The BldK complex is located at the beginning of the bald signaling cascade and is a well-known oligopeptide transporter that acts as a differentiation signal for S. coelicolor.49,50 BldG (SCO3549) is a transcriptional regulator that constitutes one of the latter steps of the bald cascade and participates in S. coelicolor sporulation.51 It has an intriguing expression pattern: it was up-regulated in the intrinsic membrane fraction of MII (4-fold in solid and 2-fold in liquid) (Table 2); however, its abundance in cytosol is reduced in MII (0.6- and 0.9-fold) (Table 2). One histidine kinase (SCO1630) was detected in the intrinsic and 4808


extrinsic membrane fractions in greater abundance in MII in solid and liquid (MII/MI ratios between 2 and 6) (Table 2). Further work will be necessary to characterize the biological significance of these proteins. In summary, 83% of all identified proteins showed similar abundance values in MI and MII in solid and liquid cultures (MII/ MI greater than or less than 1 in both conditions). MI should be considered as vegetative, since proteins governing primary metabolism were detected in greater abundance in this mycelium; MII corresponds to the differentiated Streptomyces hyphae involved in secondary metabolism synthesis, since it had greater abundance values of secondary metabolism proteins. The most remarkable differences between MII from solid and liquid cultures involved proteins regulating the final stages of hyphae compartmentalization and spore formation. These proteins were clearly down-regulated in the MIIL, which is consistent with the absence of sporulation in these conditions (whiB family sigma factor, Spo0M homologous protein or DivVA homologue protein) (see above). In addition, several putative regulatory proteins (transcriptional regulators, kinases, etc.) (see above) were detected as differentially expressed during the MI and MII stages. These

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Figure 6. Differential protein abundance values (logarithm of iTRAQ ratio) for differentially expressed proteins between MI and MII. Proteins shown were those detected in only one subcellular fraction (cytosolic or membrane) and were quantified in all developmental phases. (a) Proteins with similar abundances in liquid and solid cultures: MII/MI log iTRAQ ratios greater than 0.2 or less than -0.2 and MIL/MIS and MIIL/MIIS log iTRAQ ratios between -0.2 and 0.2. (b) Proteins with inverse logiTRAQ ratios in liquid and solid cultures: MII/MI, MIL/MIS, and MIIL/MIIS log iTRAQ ratios greater than 0.2 or less than -0.2. MI, first compartmentalized mycelium; MII, second multinucleated mycelium; S, solid cultures; L, liquid cultures. Table 2. Distribution and Abundance Values of Proteins Detected in More than One Subcellular Fractiona MIIS/MIS

function

SCO no.

function

Translation, protein folding SCO1998 30S ribosomal protein S1 Transporters/Secreted SCO7677 Secreted solute-binding protein SCO2780 Secreted protein Transcriptional regulators SCO3571 Transcriptional regulator Kinases SCO1630 Histidine kinase-like ATPases DNA/RNA replication SCO3873 DNA gyrase subunit A Bld Whi proteins SCO3549 BldG SCO5115 BldKD SCO5113 BldKB SCO5995 Secreted protein with TTA leucine codon

C

I

0.6 1.6 2.5 1

0.5 1.9 ns 2 3.7 1.2 4.8 0.8 ns

6 0.6 0.9 4

MIIL/MIL

E

C

I

MIL/MIS

E

0.2 0.6 0.7 0.3 1.6 ns 1.9 ns 1.7 3.1 ns 1.7 ns 1.9 2.2 2.6 ns 3.1 5.6 0.9 2 0.9 ns ns 0.7 0.8 0.7 0.7 1.9 2.1 1.9

C 1 ns 1.5 0.7

I

1 0.8 1 0.8 1.2 1.4 1 ns 1.1 ns 1.1 ns

MIIL/MIIS

E

C

0.6 1 1 0.8 1.2 1.9 ns 1.1 0.7 1.1 0.6 ns 1.1 1.2 0.5

I

E

1.4 0.8 1 0.8 ns 4

1 0.8 1.2

1.3 ns

0.8 ns 0.7 1.2 1.3

a Proteins included here are those detected in more than one subcellular fraction and with greater divergences between their abundance values in MI and MII from liquid and solid cultures (Figure 7) (see Supporting Information Table 4 for details). Proteins encoded by the bald developmental genes are also included. Labelling as in Table 1. Numbers in bold correspond to the abundance values included in Figure 7. Abbreviations as in Table 1.

proteins presumably regulate presporulation developmental phases and current studies are aimed toward revealing their biological functions. This work revealed that differentiation in liquid cultures is much more similar to solid cultures than might be expected in the context of the classical Streptomyces developmental model, demonstrating the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the subsequent multinucleated hyphae in liquid cultures. More detailed knowledge of the differences between the MI (vegetative) and MII (reproductive) proteomes represents a huge advance in Strepto-

myces biology and enable future analyses of specific proteins that induce, maintain, and contribute to these differences. Such experiments will advance our understanding of the biomolecular pathways controlling the presporulation differentiation phases of Streptomyces and will have implications for optimization of industry-scale fermentation processes.

Conclusion The work presented here is pioneering in approaching Streptomyces industrial fermentations from the point of view Journal of Proteome Research • Vol. 9, No. 9, 2010 4809

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Manteca et al.

References

Figure 7. Differential protein abundance values (logarithm of iTRAQ ratio) for differentially expressed proteins detected in several cellular locations. Proteins shown were those detected in more than one subcellular fraction (cytosolic or membranes); quantified in all developmental phases; with different abundances in MI and MII (MII/MI logarithm iTRAQ ratio >0.2 or

Quantitative Proteome Analysis of Streptomyces coelicolor

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