ABSTRACT
Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome.
IMPORTANCE Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.
INTRODUCTION
Turnover of cellular proteins is required in all living organisms to maintain normal cellular physiology. In eukaryotes, the ubiquitin-proteasome pathway is the major route of protein degradation and turnover (1). Ubiquitin is a 76-residue protein that can be conjugated to target proteins, marking them for degradation by the 26S proteasome complex. In addition to its role in tagging proteins for degradation, ubiquitination is also involved in protein transport and localization. It thereby contributes to regulating various cellular functions such as transcription, stress responses, apoptosis, and DNA repair (1, 2).
In bacteria, proteolysis is due to several proteases possessing an AAA+ (ATPases associated with a variety of cellular activities) domain, including ClpAP, ClpXP, Lon, HslUV, and FtsH (3, 4). A bacterial 20S proteasome has also been described for some bacteria, including actinobacteria such as Rhodococcus erythropolis (5), Streptomyces coelicolor (6), Frankia alni (7), Mycobacterium smegmatis (8), and Mycobacterium tuberculosis (9). Unlike its eukaryotic counterpart, the actinobacterial proteasome is not essential (8–11), although its inactivation can be associated with various deleterious effects. For example, proteasome inactivation in M. tuberculosis impairs growth on agar media and reduces virulence in mice (9, 10). Conversely, proteasome mutants of Streptomyces lividans and M. smegmatis were described to be phenotypically indistinguishable from the wild-type strains under both normal and stress-inducing conditions (8, 11). Proteasome inactivation in S. coelicolor has only a modest effect on cell physiology: it was reported to increase resistance to cumene hydroperoxide, an effect associated with increased levels of a haloperoxidase enzyme (12). In addition, recent results suggest that the proteasome in S. coelicolor has a role in cell differentiation: proteasome-deficient mutants produce smaller amounts of pigmented antibiotics than the wild type and are delayed in their morphological development (13).
The roles of these bacterial proteasomes were unclear until a protein-tagging system specifically directing proteins for proteasomal degradation was discovered in mycobacteria (14, 15). This ATP-dependent pathway shares many functional similarities with the eukaryotic ubiquitin pathway (16). However, the sequence and structure of the modifier peptide, named Pup (prokaryotic ubiquitin-like protein), as well as the conjugation process differ from those of the ubiquitin pathway (16–20). In M. tuberculosis and M. smegmatis, Pup possesses a conserved C-terminal GGQ motif that requires deamidation of glutamine to glutamate by Dop (deamidase of Pup). PafA (proteasome accessory factor A) catalyzes isopeptide bond formation between the C-terminal glutamate of Pup and the ε-amino group of a lysine in the target protein (4).The pupylated protein is recognized and unfolded by Mpa (Mycobacterium proteasomal ATPase) and then degraded in the proteasome (21–23). Dop is also able to remove Pup from pupylated proteins, a process named depupylation (18, 24, 25).
Following the discovery of pupylation, several proteome-wide studies reported the identity of Pup-tagged proteins (pupylome) in mycobacteria (26–28). Pupylation was also demonstrated in Rhodococcus erythropolis (29) and in Corynebacterium glutamicum (30), and proteins targeted by pupylation in these organisms have been identified. Pupylated targets are numerous and belong to various functional classes of proteins. The mycobacterial genes pafA and pup (gene modified to encode a protein with a GGE C terminus) have been expressed in Escherichia coli, resulting in the pupylation of host proteins (31). This provides clear evidence that no mycobacterial cofactor is essential for substrate recognition by PafA.
To date, pupylation has been studied mainly in mycobacteria. Nevertheless, several actinobacteria, including streptomycetes, carry orthologues of pup, dop, and pafA that are likely to be involved in pupylation (14, 32). Streptomyces species are filamentous bacteria that undergo complex morphological differentiation and produce a great variety of secondary metabolites, including antibiotics, with important applications in human medicine and in agriculture. Both secondary metabolism and morphological differentiation are controlled by an intricate regulatory network; this network has been extensively studied in the reference strain of Streptomyces coelicolor (33, 34). Proteolysis is one of the mechanisms by which the differentiation of Streptomyces is regulated. For example, deficiency in the ATP-dependent Clp protease in S. lividans alters morphological differentiation (35), and BldD and SigT, key regulators of Streptomyces development, are subject to specific proteolytic control (13, 36). These various observations led us to study pupylation in S. coelicolor.
Here, we present an investigation of pupylation in S. coelicolor, including analyses of the transcription of genes involved in pupylation and the identification of some pupylated substrates. We also studied the effects of pup deletion particularly on morphological differentiation, secondary metabolism, and resistance to oxidative stress. We demonstrate that pupylation plays an important role in both sporulation and antibiotic production, revealing an additional level of developmental regulation in S. coelicolor. The phenotypes of pup and proteasome mutants suggest that pupylation is part of normal cell physiology through both proteasome-dependent and -independent actions.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions.Table S1 in the supplemental material lists strains and plasmids used in this work. S. coelicolor A3(2) strain M145 (wild type) and mutant strains were grown on standard media and manipulated according to well-established protocols (37). Escherichia coli strains were grown in LB medium. E. coli DH5α was used as a host for plasmid constructions according to standard procedures (38). E. coli ET12567/pUZ8002 was used for conjugation of shuttle vectors from E. coli to S. coelicolor (37).
Construction of a S. coelicolor proteasome mutant by prcB disruption.Table S2 in the supplemental material lists the oligonucleotides used in this work. A proteasome-deficient strain was constructed by disrupting the prcB gene. An internal fragment of prcB (557 nucleotides [nt] of the 846-nt prcB coding sequence) was amplified (with primers HBP88 and HBP89) and ligated into the pGEM-T Easy vector. The EcoRI-prcB-EcoRI fragment was obtained by digestion with EcoRI and inserted into the single EcoRI site of pOJ260, yielding pOJ260-prcB. This construct was transferred from E. coli to S. coelicolor by conjugation. The S. coelicolor prcB disruption mutant was selected for apramycin resistance and analyzed by appropriate PCR amplifications, with verification by sequencing. We showed, by reverse transcription-PCR (RT-PCR), that the insertion into prcB abolished prcA transcription (data not shown), as expected, if the two genes were cotranscribed. We checked the stability of this mutant by culturing it for 5 days without selective pressure. We tested 500 clones from this culture, all of which were found to be resistant to apramycin.
Construction of a S. coelicolor pup deletion mutant.Upstream and downstream regions of pup were amplified from S. coelicolor genomic DNA by PCR and inserted into pGEM-T Easy (yielding pGEM-T-Easy-pupBG and pGEM-T-Easy-pupBD). We checked for an absence of mutations in the up- and downstream fragments by sequencing. The upstream and downstream fragments were inserted, together with the apramycin resistance cassette att3-aac (39), into the suicide vector pOSV400 (carrying a hygromycin resistance gene), yielding pOSV400-pup. In this plasmid, the pup upstream and downstream fragments flank the apramycin resistance gene. The pOSV400-pup plasmid was introduced into S. coelicolor by conjugation. S. coelicolor transconjugants resistant to apramycin were selected and screened for hygromycin sensitivity. The replacement of the pup gene with the apramycin resistance cassette was checked by PCR amplification (primers HBP5/L-acc2R and R-acc2F/HBP6). The apramycin resistance cassette was then excised by site-specific recombination, as previously described (39). The resulting pup locus was then amplified by PCR and sequenced (with the HBP5/HBP6 primers) to confirm that 206 bp had been deleted from the pup coding sequence and replaced with a 41-bp sequence.
Construct for the expression of His6-Pup.The coding sequence of pup was amplified from S. coelicolor genomic DNA (see Table S2 in the supplemental material for primers). The pSET-E*-His-pup construct was obtained by amplification with primers NS9 and NS12, generating an NdeI-His-pup-BamHI fragment. Primer NS9 was used to introduce a sequence encoding a hexahistidine tag upstream from the modified pup start codon. The NdeI-His-pup-BamHI fragment was inserted between the NdeI/BamHI sites in pHM11a such that the pup gene was under the control of the ermE* promoter. The ermE*-His-pup fragment was excised and inserted into the integrative vector pSET152 (XbaI/BamHI sites); the resulting plasmid was introduced into S. coelicolor wild-type or Δpup strains by conjugation (37).
SDS-PAGE and immunoblot analysis.Protein samples were prepared from 12 ml of cultures grown at 30°C in yeast extract-malt extract (YEME) or R2YE (37) liquid medium for 45 h. The mycelium was ground with glass beads (size, <106 μm; Sigma-Aldrich) in a FastPrep shaker (40 s at a 4.5 intensity; Q-BIOgene), and the extracts were clarified by centrifugation (10 min at 12,000 rpm). Protein concentrations in supernatants were determined by using the Bio-Rad protein assay, and samples were treated with Laemmli buffer and XT reducing agent (Bio-Rad).
SDS-PAGE was performed by using Criterion XT Bis-Tris precast 12% gels (Bio-Rad) with a Criterion electrophoresis system (Bio-Rad). Molecular size reference markers were obtained from Bio-Rad.
For immunoblot experiments, proteins were transferred from the gel to Hybond ECL nitrocellulose membranes by using a semidry transfer system (Bio-Rad). Immunodetection was based on Penta-His horseradish peroxidase (HRP) conjugate antibodies (Qiagen) and the Pierce ECL Western blotting substrate (Thermo Scientific), according to the manufacturers' protocols.
Preparation of pupylated targets.Strains were grown in 500 ml YEME (5 mM MgCl2) or R2YE liquid medium for 45 h in baffled Erlenmeyer flasks. The mycelium was harvested by centrifugation and washed once with 50 ml of buffer I (50 mM NaPO4, 300 mM NaCl, 20 mM imidazole [pH 8]). The mycelium was then suspended in buffer I (25 ml) and disrupted with a French press. Cell debris was removed by centrifugation, and the supernatant was filtered through 0.2-μm-pore-size membranes (Millipore). Crude extracts were then loaded onto a 0.3-ml Ni-nitrilotriacetic acid (NTA)-agarose column (Qiagen) previously equilibrated with buffer I, and the column was washed with buffer II (50 mM NaPO4, 300 mM NaCl, 30 mM imidazole [pH 6.5]). The proteins were eluted with an imidazole gradient (30 to 500 mM) and recovered in 300-μl fractions. Elution was monitored by using a Uvicord SII UV detector (Pharmacia), and fractions were analyzed by SDS-PAGE and Coomassie blue staining. Selected fractions were pooled in a Slide-A-Lyzer cassette with a 3,500-molecular-weight cutoff (MWCO) (Thermo Scientific), dialyzed twice with 1 liter of ammonium acetate (300 mM, pH 7), and then dried in a Speed-Vac concentrator.
Mass spectrometry analysis, database searches, and protein identification.Dried protein fractions were reconstituted in a solution containing 100 mM Tris HCl (pH 8.5) and 8 M urea, reduced [5 mM Tris(2-carboxyethyl)phosphine], and alkylated (10 mM iodoacetamide). Endoproteinase Lys-C (Promega, Madison, WI, USA) was added at a 1:40 (wt/wt) dilution. The sample was incubated for 5 h at 37°C and then diluted 1:4 with 100 mM Tris HCl (pH 8.5). Trypsin Gold (mass spectrometry [MS] grade; Promega, Madison, WI, USA) was added at a 1:100 (wt/wt) dilution, and the digestion was allowed to proceed overnight at 37°C with shaking. The same quantity of enzyme was then added, and the sample was incubated for 5 h at 37°C with shaking. The sample was desalted with Omix C18 solid-phase extraction pipette tips (Varian), dried, and reconstituted in H2O-acetonitrile-formic acid (98:2:0.1).
The resulting trypsin-digested peptides were analyzed by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 system (Dionex, Amsterdam, Netherlands) coupled to an LTQ-Orbitrap Velos instrument. Aliquots of 5 μl of each sample were loaded onto a C18 precolumn (300-μm inner diameter by 5 mm; Dionex) at 30 μl/min in a solution containing 2% ACN and 0.1% FA. After 5 min of desalting, the precolumn was switched online with a 3-μm analytical PepMap100 C18 column (Dionex) equilibrated in 95% solvent A (2% ACN, 0.1% FA) and 5% solvent B (80% ACN, 0.08% FA). The peptides were eluted with a 2 to 60% gradient of solvent B over 37 min at a flow rate of 300 nl/min. The LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) was operated in the data-dependent acquisition mode with XCalibur software (Thermo Fisher Scientific, Bremen, Germany). Survey scan mass spectra were acquired with the Orbitrap instrument in the range of 300 to 2,000 m/z with the resolution set to a value of 60,000 at m/z 400. The 20 most intense ions per survey scan were selected for collision-induced dissociation (CID) fragmentation, and the resulting fragments were analyzed in the linear trap (LTQ). Dynamic exclusion was employed within 20 s and repeated after 30s to prevent repetitive selection of the same peptide.
The Mascot v.2.4.1 search engine was used with Proteome Discoverer version 1.4.0.288 (Thermo Fisher Scientific, Bremen, Germany) to search a database of concatenated S. coelicolor proteins (Swiss-Prot) and known contaminants and reversed sequences of all entries (8,286 entries in total). The following search parameters were applied: carbamidomethylation of cysteines was set as a fixed modification, and pupylation of lysine (+243.086 Da due to the addition of a deamidated QGG motif [15]), oxidation of methionine, and carbamylation of lysine were set as variable modifications. The specificity of trypsin digestion was set for cleavage after Lys or Arg, and two missed cleavage sites were allowed. The mass tolerances for MS and MS/MS were set to 10 ppm and 0.5 Da, respectively. Scaffold software (version Scaffold_3.5.1; Proteome Software Inc., Portland, OR) was used for probability assignment and validation.
The raw data files were converted into mgf format by using Proteome Discoverer and analyzed with the InSpecT open-source search engine (http://proteomics2.ucsd.edu/LiveSearch/index.jsp), which was developed at the UCSD NIH NCRR center for computational mass spectrometry. The searches were performed with the same information for enzymes and modifications.
Scanning electron microscopy (SEM).Streptomyces strains were cultured for 5 days on a cellophane membrane placed on top of solid agar medium. Strips (4 cm by 2 cm) of the cellophane with well-developed mycelium growth were cut out and mounted onto glass slides, attached by Scotch tape. The slides were placed into a desiccator with a small container of 2% OsO4 in double-distilled water (ddH2O) and left for the mycelia to be fixed in osmium vapor for several days. Fixed and air-dried samples were sputter coated with gold using a Polaron SC510 sputter coater (Quorum Technologies Ltd., Lewes, United Kingdom) and examined with an Aquasem scanning electron microscope (Tescan, Brno, Czech Republic) in high-vacuum mode. The images of mycelia were randomly taken at both the periphery and central parts of the mycelium. Selected samples were reexamined at higher resolution with a Tescan Vega LSU scanning electron microscope (Tescan, Brno, Czech Republic) at 20 kV.
Determination of secondary metabolite production.S. coelicolor produces several secondary metabolites, including blue-pigmented actinorhodin (ACT), red-pigmented prodiginines (RED), and nonpigmented calcium-dependent antibiotic (CDA) (37). ACT and RED were assayed in R2YE liquid medium (40). About 1010 spores were used to inoculate 50 ml of R2YE medium in a 250-ml baffled flask. The cultures were grown at 30°C with shaking at 180 rpm. Samples of 1 ml were taken for RED and ACT assays. The samples were centrifuged for 10 min; both the supernatant and the pellet (the mycelium) were used for ACT assays, whereas only the mycelium was used for RED assays (40). The production of CDA on Oxoid nutrient agar medium was evaluated by a bioassay using Micrococcus luteus as the indicator (41).
Tests for resistance to H2O2.Each strain was grown in liquid tryptic soy broth (TSB) medium. The mycelium was harvested, and the same weight of mycelium from each strain was used. Mycelial pellets were disrupted by repeated passage through a needle (21 gauge). The optical density of each sample was determined, to allow all inocula used to be equal. The inocula were added to 3.5 ml of liquid soft nutrient agar (SNA) (37); this was poured onto R2YE plates for agar diffusion assays. A 4-mm-diameter cylinder was removed from the center of each plate, and 100 μl of 30% (8.8 M) H2O2 was poured into the hole. Results were read after 5 days of growth at 30°C. The quantitative assay involved spreading of dilutions of the disrupted mycelium onto R2YE medium supplemented or not with 8.8 mM H2O2. Colonies were counted after 5 days of growth.
RESULTS
Expression of pupylation and proteasome genes in S. coelicolor.The actinobacterial 20S proteasome consists of a proteolytic compartment assembled from the PrcA and PrcB proteins. Another protein, called Arc (for AAA+ ATPase forming ring-shaped complexes) or Mpa (mycobacterial proteasomal ATPase) in mycobacteria, is also associated with this proteasome. Orthologues of the mycobacterial genes involved in pupylation (pup, dop, and pafA) are present in the genomes of Streptomyces spp., in which they are clustered with genes encoding components of the proteasome (arc, prcA, and prcB) (Fig. 1A). The organization of the locus is mostly conserved among streptomycetes. The products of the pupylation and proteasome genes in S. coelicolor are similar (70 to 92% amino acid similarity) to those in mycobacteria. However, there is a marked difference in that the Pup protein in S. coelicolor has a C-terminal glutamate residue, whereas the corresponding position in the mycobacterial protein is occupied by a glutamine residue (Fig. 1B). Dop is therefore not required for the deamidation of S. coelicolor Pup before its linkage to a target protein. The conservation of Dop in S. coelicolor might be explained by its possible role in depupylation, as shown in Mycobacterium (18).
Pupylation and proteasome genes from mycobacteria and streptomycetes. (A) Comparison of the regions containing the pupylation and proteasome genes in Mycobacterium tuberculosis (Mt), Mycobacterium smegmatis (Ms), Streptomyces coelicolor (Sc), Streptomyces avermitilis (Sa), and Streptomyces griseus (Sg). Genes represented by arrows of the same color are orthologues, except those represented by white arrows, which are not syntenic in actinobacteria. The only difference between Streptomyces strains is the presence, in some strains, of a gene encoding a putative endonuclease VII (SCO1645 in S. coelicolor and SAV_6680 in S. avermitilis). The proteins SCO1641 (a putative transporter of the MFS family), SCO1642 (a putative LacI-like repressor), and SCO1645, and their orthologues in other Streptomyces species, are not predicted to be involved in functions related to pupylation or proteasomal degradation. (B) Alignment of Pup proteins from these five species.
We studied the transcription of the pupylation (pup, dop, and pafA) and proteasome (prcA, prcB, and arc) genes during S. coelicolor development by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (see Fig. S1 in the supplemental material). Specific transcripts of all six genes were detected at each of the time points tested (24 h, 48 h, and 72 h). We also made use of some of the transcriptomic data sets available for S. coelicolor M145 in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) (42) (see the supplemental material). This analysis confirmed the transcription of the pupylation and proteasome genes at various stages of growth and development and showed that they were among the 5% of genes with the highest transcription levels. In S. coelicolor, the pup, dop, prcB, and prcA genes are in the same orientation, separated by only short intergenic regions or even overlapping (e.g., SCO1645-prcB), suggesting possible cotranscription. Indeed, the cotranscription of these genes was predicted by an in silico analysis (43). An analysis of the transcriptome sequencing (RNA-seq) data sets available from the GEO showed that many reads overlapped two consecutive genes in this region, suggesting that these genes are indeed cotranscribed (see the supplemental material). A semiquantitative analysis of protein abundance was performed by Thomas et al. for S. coelicolor M145 (44). The results obtained showed that Arc, Dop, PcrA, PcrB, and PafA were among 650 proteins detected in all time courses and that these proteins were relatively abundant.
Pupylation in S. coelicolor.We searched for Pup-tagged proteins in S. coelicolor. An N-terminally His6-tagged version of Pup was produced in S. coelicolor, allowing subsequent detection of pupylated proteins with anti-His6 antibodies. As pup was expressed at different stages of S. coelicolor growth and development, the gene encoding His6-Pup was cloned under the control of the constitutive promoter ermE* (45); the construct was inserted into the integrative vector pSET152, yielding pSET-E*-His-pup. This construct complemented the pup deletion mutant (see below). The wild-type S. coelicolor strain and strains harboring pSET152 or pSET-E*-His-pup were each grown in two different liquid media (YEME and R2YE) for 45 h. Protein samples were prepared from each of these cultures and analyzed by immunoblotting using an anti-His6 antibody (Fig. 2). Many signals were observed with the extracts of the strain expressing His6-Pup grown in both media. No or few detectable signals were observed under the same experimental conditions for samples from either the wild type or the strain harboring the empty vector. The few signals observed in these control samples may have been due to histidine-rich proteins. These experiments indicated that protein pupylation occurs in S. coelicolor and that an apparently large number of proteins are targeted by this system.
Immunoblots for the detection of pupylated proteins with anti-His6 antibodies. (A) Proteins extracted from mycelium grown in liquid R2YE medium for the wild-type strain (lane 1), the wild-type strain harboring the empty vector pSET152 (lane 2), and the wild-type strain harboring pSET-E*-His-pup (lane 3). (B) Proteins extracted from mycelium grown in liquid YEME medium for the wild-type strain (lane 1), the wild-type strain harboring the empty vector pSET152 (lane 2), and the wild-type strain harboring pSET-E*-His-pup with the same exposure time as that for lanes 1 and 2 (lane 3) and a shorter exposure time (lane 4).
Identification of pupylated proteins in S. coelicolor.We undertook a proteome-wide analysis of pupylated proteins in S. coelicolor to check that Pup bound covalently to lysine residues and to identify some of the proteins tagged. Our analysis was based on previous studies in mycobacteria showing the binding of a GGE tripeptide tag to lysine residues in tryptic digests of pupylated proteins, associated with a +243-Da shift (26–28). The C-terminal sequence of the S. coelicolor Pup is KGGE, so we expected pupylation to lead to the same mass shift for trypsin-digested pupylated peptides. For this proteome-wide study, we used S. coelicolor strains (wild type and a pup deletion mutant) harboring the pSET-E*-His-pup vector. In these strains, His6-tagged Pup was linked to a large number of proteins. These S. coelicolor strains grown in two different liquid media (YEME and R2YE) presented two different profiles on immunoblots with the anti-His6 antibody (Fig. 2). We therefore studied the pupylomes for each of these two media. Ni-NTA affinity chromatography was used to enrich cell lysates in His-tagged pupylated proteins. The eluted fractions containing tagged proteins were then pooled for each culture and digested with trypsin. The resulting peptides were analyzed by LC-MS/MS. Proteins were identified by at least two unique peptides with probability scores of >95%.
We identified 467 proteins in the sample from the wild-type strain harboring pSET-E*-His-pup grown in YEME medium and 1,579 proteins in the sample from the pup deletion mutant strain harboring pSET-E*-His-pup grown in R2YE medium. A +243-Da modification, which could be attributed to the presence of the GGE tag on a lysine residue, was detected on 23 peptides, corresponding to 20 proteins (Table 1). Both Mascot and InSpecT proteomic search engines were used for these analyses. The pupylation tag was confirmed by a detailed inspection of the spectra of the peptides. The number of proteins with identifiable pupylation sites was small, possibly because Dop may be copurified with the pupylome, leading to the depupylation of some proteins, as previously reported (24).
Pupylated proteins identified in S. coelicolor
Three pupylated proteins (GroEL2, AhpC, and SCO4496) were detected in both media. Six pupylated proteins were detected in the YEME pupylome, and 11 were detected in the R2YE pupylome. The 20 pupylation targets identified belong to 11 of the 23 Cluster of Orthologous Groups (COG) functional groups (Table 1). The functional group most frequently identified was posttranslational modification, protein turnover, and chaperone functions (O); four of the pupylation target proteins belonged to this group. Orthologues of seven of the 20 proteins (AcpP, FabF, PurA, Tuf1, GroEL1, AhpC, and SCO6042) were also identified in at least one of the pupylomes reported for M. tuberculosis, M. smegmatis, R. erythropolis, or C. glutamicum (26–30). However, 10 proteins pupylated in S. coelicolor were not described in any of the mycobacterial, Rhodococcus, and Corynebacterium pupylome analyses despite their conservation in these genera. SCO6042 and SCO3629 were the only proteins pupylated in S. coelicolor for which homologous proteins were also found to be pupylated in three of the four other actinobacterial species (Table 1; see also Fig. S2 in the supplemental material). For SCO6042, the same lysine was pupylated in S. coelicolor and R. erythropolis, but a different lysine was found to be pupylated in the two mycobacteria. For SCO3629, the lysine pupylated in S. coelicolor was found to be conserved in the three other actinobacteria, but a different lysine was pupylated in these other strains.
Morphological differentiation of pupylation- and proteasome-deficient mutants.Proteolysis is linked to the control of Streptomyces differentiation (35, 36, 46). The pupylation-directed degradation of proteins by the 20S proteasome may therefore be involved in differentiation. We investigated the role of pupylation in S. coelicolor by inactivating genes encoding Pup or components of the 20S proteasome. A pup mutant was constructed, in which most of the pup coding sequence was deleted. A proteasome-deficient mutant was constructed by disrupting prcB by the insertion of pOJ260, conferring apramycin resistance. This insertion abolished the transcription of prcA (data not shown) but should have no effect on the expression of genes other than prcB and prcA, as the gene downstream from prcA is in the opposite orientation (Fig. 1).
The wild-type strain and the pup and proteasome mutants were grown on solid soy flour-mannitol (SFM) medium (37) and the colonies obtained were compared in terms of their appearance. After 5 days of growth, the mycelium of the pup deletion mutant was much less pigmented than that of the wild-type strain (Fig. 3). The medium surrounding the pup deletion strain was darker than that surrounding the wild-type strain, suggesting a higher level of production of pigmented secondary metabolites. The wild-type phenotype was restored in the mutant strain by transformation with pSET-E*-His-pup. No such complementation was observed with the empty vector (pSET152). We can therefore conclude that complementation was due to pup expression. The growth and differentiation of the proteasome-deficient mutant on SFM agar did not appear to be different from those of the wild type (Fig. 3B). We monitored the appearance of the mycelium of each strain over a period of 10 days. The phenotype of the pup mutant remained unchanged throughout this longer incubation (see Fig. S3 in the supplemental material). The differences observed were therefore due to a defect in differentiation and not to a delay in the differentiation of the pup mutant.
Morphological differentiation on solid SFM (A and B) and R2YE (C and D) media. The wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup+pSET152), the pup mutant with pSET-E*-His-pup (Δpup+pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260) are shown after 5 days of growth.
A second medium, R2YE, a rich medium also used to visualize the effects of mutation on Streptomyces differentiation (34, 47), was also used to study the effects of pupylation and proteasome defects. Again, the mycelia of the five strains grown on solid R2YE medium were examined by eye (Fig. 3D). The pup deletion mutant was clearly different from the wild-type strain, with a much less pigmented aerial mycelium and a lower level of production of pigmented secondary metabolites. The proteasome-deficient mutant appeared to be almost unaffected, displaying only a slightly lower level of mycelium pigmentation than the wild-type strain. In the pup deletion mutant, a phenotype similar to that of the wild type was restored by the expression of His-tagged Pup. This restoration could clearly be attributed to Pup expression, as it was not observed for the pup deletion mutant containing the empty vector.
Differences in aerial mycelium pigmentation may be linked to sporulation defects (48–50). Consistent with this hypothesis, we found that the yield of viable spores from the pup deletion strain in confluent cultures on R2YE or SFM plates was ∼1% that of the wild type (data not shown), suggesting that spore formation was impaired.
The differences in colony morphology upon visual examination and in viable spore counts led us to use scanning electron microscopy (SEM) to evaluate aerial mycelia and spore formation in these strains. After 5 days of growth on solid R2YE medium, we harvested samples from each of the five strains and prepared them for SEM (Fig. 4). The wild-type strain displayed three dominant features: (i) the aerial mycelium was abundant, (ii) many spore chains were visible, and (iii) the different stages of spore development were represented as visible immature and mature spore chains. The pup deletion mutant also had an abundant aerial mycelium, but many of the hyphae were collapsed and thin, and few of the typical spore chains were observed. In the pup deletion mutant transformed with the empty pSET152 vector, spore chains were even scarcer. Complementation with the pup gene under the control of the constitutive ermE* promoter (pSET-E*-His-pup) restored a phenotype similar to that of the wild-type strain. The proteasome-deficient mutant displayed impaired aerial mycelium development on R2YE medium (Fig. 4).
Scanning electron microscopy of mycelium grown on R2YE medium. Scanning electron micrographs of surface cultures grown on R2YE medium for 5 days are shown for the wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup+pSET152), the pup mutant with pSET-E*-His-pup (Δpup+pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260). The same magnification is used for all images. Bar, 10 μm.
On SFM medium, the pup deletion mutant also displayed phenotypic impairment, with far fewer coils and spore chains than the wild type (see Fig. S4 in the supplemental material). The expression of His-Pup restored normal sporulation. In contrast to the results obtained with R2YE medium, the proteasome-deficient mutant presented normal aerial mycelium development and sporulation, highlighting the existence of a medium-dependent phenotype in this mutant (see Fig. S4 in the supplemental material).
Production of secondary metabolites by pupylation- and proteasome-deficient mutants.The production of pigmented secondary metabolites by the pup-deficient mutant on R2YE medium differed from that of the wild-type strain. We therefore studied the involvement of the pupylation and proteasome systems in antibiotic production. Under typical laboratory conditions, S. coelicolor produces two pigmented antibiotics, RED and ACT, together with the nonpigmented CDA (37). We tested the effect of the absence of pupylation or of the proteasome on the production of these antibiotics. For CDA, we observed no significant difference in the diameters of the inhibition zones surrounding the colonies of the five strains challenged with Micrococcus luteus on a plate (see Fig. S5 in the supplemental material).
ACT and RED production levels were monitored over 7 days of growth in liquid R2YE medium (Fig. 5). The levels of both ACT and RED produced by the pup mutant (with or without the empty vector) were substantially lower than those of the wild-type strain over the entire 7-day period. The production of His-Pup in the pup mutant restored ACT production to levels similar to those in the wild-type strain. For RED production, the restoration was only partial. For the proteasome mutant, only ACT production differed from that of the wild-type strain, and the difference was modest (the mutant produced half the amount produced by the wild-type strain).
Time courses of actinorhodin (ACT) and prodiginine (RED) production in liquid R2YE medium. The production of ACT and RED is shown for the wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup + pSET152), the pup mutant with pSET-E*-His-pup (Δpup + pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260).
Sensitivity of pupylation- and proteasome-deficient mutants to oxidative stress.Oxidative stress has been linked to pupylation and the proteasome in both Mycobacterium (9, 10, 18, 51, 52) and Streptomyces (12). We therefore also assessed the sensitivity of each of the five S. coelicolor strains to oxidative stress. Hydrogen peroxide was used as an oxidant in solid R2YE and SFM media. We first observed the inhibition of growth for each strain following exposure to a gradient of concentrations of hydrogen peroxide in an agar diffusion test. The pup deletion mutants presented a halo of inhibition larger than those of the other strains on R2YE medium (Fig. 6A) and on SFM medium (data not shown). We then evaluated the effects of oxidative stress by plating disrupted mycelia from the five strains onto R2YE solid medium with and without hydrogen peroxide. Colonies were counted after 5 days of growth (Fig. 6B). The pup deletion mutant (with or without the empty vector) was much more sensitive to hydrogen peroxide than the other strains. This sensitivity of the pup mutant to hydrogen peroxide was complemented by introducing a copy of the His-pup gene on pSET152. The sensitivity of the proteasome mutant to H2O2 was similar to that of the wild-type strain (Fig. 6B).
Response to oxidative stress. The wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup + pSET152), the pup mutant with pSET-E*-His-pup (Δpup + pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260) were exposed to oxidative stress caused by H2O2 on R2YE medium. (A) Diffusion assay showing the inhibition zone caused by H2O2. (B) CFU counts on medium with or without H2O2.
DISCUSSION
Our objectives were to determine whether pupylation occurs in Streptomyces and, if so, to investigate its role in morphological and physiological differentiation, comparing the results obtained with those for the proteasome. We chose to perform the study with S. coelicolor, the model organism used for most studies of development in filamentous actinobacteria. In streptomycetes, as in mycobacteria, the genes involved in pupylation and those encoding components of the proteasome are clustered together on the chromosome. In S. coelicolor, these genes were transcribed at various stages of development on solid medium and at all time points tested in liquid culture. This finding is consistent with data from previous proteome studies in S. coelicolor, which have shown that all the proteins of the proteasome and the pupylation process are present at various times during growth, including the early stages (44).
We then demonstrated that the pupylation process was active in S. coelicolor and that numerous proteins were modified by this process. We identified 20 pupylation targets in S. coelicolor. Seven of these target proteins have homologues that are also pupylated in Mycobacterium, R. erythropolis, or C. glutamicum (26–30). These homologous proteins were, in some cases, modified at different positions in different actinobacteria (see Fig. S2 in the supplemental material). As a result, the currently available prediction programs based on the mycobacterial pupylome (53–55) do not predict pupylation sites in S. coelicolor proteins accurately. Indeed, the three available prediction programs predicted only 6 to 11 of the 22 modified lysine residues identified in S. coelicolor pupylated proteins (see Table S3 in the supplemental material). One of the 20 pupylated proteins identified in this study, AhpC (SCO0465), has already been shown to accumulate in a S. coelicolor proteasome-deficient mutant and in arc, dop, and pup insertion mutants (12). These observations are consistent with the degradation of pupylated proteins by the proteasome.
Investigations of the consequences of pup deletion for morphological differentiation showed that the pup mutant displayed a major impairment of spore formation on R2YE and SFM media. On the contrary, for the proteasome mutant, differentiation was not affected on SFM medium, but spore formation was reduced on R2YE medium. The pup mutant produced significantly less of the secondary metabolites ACT and RED than the wild type in liquid R2YE medium. For the proteasome-deficient mutant, the production of ACT and RED was not affected (e.g., production of ACT in liquid R2YE medium) or was only slightly affected (e.g., halving of the production of RED in liquid R2YE medium). Mao and coworkers also observed that a proteasome-deficient mutant grown on solid R2YE medium produced smaller amounts of pigmented secondary metabolites than the wild-type strain (13). Consistent with our results, the preliminary data of Liu and coworkers (56) led them to conclude that pupylation was involved in the regulation of antibiotic production in S. coelicolor. Some of the proteins that we identified as pupylation targets in S. coelicolor play a role in regulating morphological differentiation or antibiotic production. This is the case, for instance, for OsaA (57) and FtsI (58), but further studies are required to link the phenotype of the pupylation-deficient mutant for morphological and metabolic differentiation with the fate of particular target proteins.
Previous studies have established a link between oxidative stress, pupylation, and proteasomes in both Mycobacterium (9, 10, 18, 51, 52) and Streptomyces (12). We therefore compared the resistance of the pupylation- and proteasome-deficient mutants to oxidative stress with that of the wild-type strain. We found that the pup deletion mutant was much more sensitive than the wild type to hydrogen peroxide on R2YE and SFM media. Under the same conditions, the sensitivity of the proteasome-deficient mutant to hydrogen peroxide was similar to that of the wild type. In a previous study (12), De Mot and coworkers studied the sensitivities of a S. coelicolor proteasome-deficient mutant and arc, dop, and pup knockout mutants to oxidative agents. They used an agar diffusion test on TSB medium. Under these conditions, those researchers observed no difference in sensitivity to hydrogen peroxide (at concentrations of up to 0.5 M). There are several possible reasons for the differences between our observations for the pup mutant and those of the previous study. For example, the medium used was not the same, and the concentration of hydrogen peroxide used in the agar diffusion test was much higher in our case (8.8 M versus 0.5 M). Our results were confirmed by a CFU assay assessing sensitivity to hydrogen peroxide.
De Mot and coworkers observed greater resistance to cumene hydroperoxide on TSB medium in an agar diffusion system for a S. coelicolor proteasome-deficient mutant and for arc, dop, and pup knockout mutants (12). In M. tuberculosis, the pupylation and proteasome mutants were found to be more resistant to hydrogen peroxide than the wild-type strain but more sensitive to other forms of oxidative stress (9, 10). The different patterns of behavior observed for the mutants of different bacteria exposed to hydrogen peroxide or cumene hydroperoxide suggest that different defense systems are active against specific classes of exogenous peroxides, as previously observed (12, 59).
Finally, the phenotype of the S. coelicolor pup deletion mutant reveals the involvement of pupylation in the regulation of Streptomyces differentiation and resistance to oxidative stress. Our results are consistent with those of Compton et al., who found that a S. coelicolor pafA-null strain displayed defects of both sporulation and secondary metabolism and who also presented evidence linking pupylation and the oxidative stress response in S. coelicolor (60). The differences in the phenotypes of the pup and proteasome mutants observed in our study indicate that pupylation may have other roles in addition to its known proteasome-associated function. This conclusion is consistent with previous observations (26, 29) that not all pupylated proteins are degraded. Furthermore, pupylation occurs in corynebacteria, which do not have a proteasome (30). Thus, in addition to targeting proteins to the proteasome for turnover, pupylation, like ubiquitination, is probably also involved in other functions.
ACKNOWLEDGMENTS
We thank Celine Adam for skillful technical assistance and Audrey Dubost, Aude Herrera, and Nicole Alloisio for assistance with statistical and bioinformatics analyses. We also thank Sylvie Lautru, Antonio Ruzzini, and Erik Vijgenboom for critical reading of the manuscript.
This work was partly supported by the European program ERA-IB (contract EIB-08-013). Funding for LTQ-Orbitrap Velos acquisition was secured through a DIM Malinf grant from the region Ile-de-France. Access to instrumental and other facilities was also obtained with European Union support (Operational Program Prague, competitiveness project CZ.2.16/3.1.00/24023) and IMIC institutional research concept RVO61388971.
FOOTNOTES
- Received 16 July 2015.
- Accepted 10 August 2015.
- Accepted manuscript posted online 17 August 2015.
- Address correspondence to Hasna Boubakri, hasna.boubakri{at}univ-lyon1.fr, or Jean-Luc Pernodet, jean-luc.pernodet{at}i2bc.paris-saclay.fr.
↵* Present address: Hasna Boubakri, Université Lyon 1, CNRS, UMR 5557, INRA, USC 1364, Ecologie Microbienne, Villeurbanne, France; Nicolas Seghezzi, Department of Microbiology and Immunology, University of British Columbia, Life Sciences Centre, Vancouver, British Columbia, Canada.
H.B. and N.S. contributed equally to this work.
Citation Boubakri H, Seghezzi N, Duchateau M, Gominet M, Kofroňová O, Benada O, Mazodier P, Pernodet J-L. 2015. The absence of pupylation (prokaryotic ubiquitin-like protein modification) affects morphological and physiological differentiation in Streptomyces coelicolor. J Bacteriol 197:3388–3399. doi:10.1128/JB.00591-15.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00591-15.
REFERENCES
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