Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus

The small γ -butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomic analysis that many nutrient scavenging and stress-related proteins are overexpressed in an A-factor nonproducing mutant (AFN) of Streptomyces griseus B2682. Trancript analysis shows that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress, environmental stress and an orthologue of the Bacillus sporulation control protein SpoOM. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression is restored to AFN by the signaling protein Factor C, in line with our earlier observation that Factor C triggers A-factor production.


INTRODUCTION
Bacteria of the Gram-positive filamentous Streptomyces are a well-known model system for the study of prokaryotic multicellular differentiation. They have a complex mycelial life cycle starting with a vegetative mycelium that develops into aerial mycelium, which then produces chains of spores at the ends of the hyphae (1). The onset of development is triggered by nutritional signals (2) and temporally relates to the production of antibiotics and other secondary metabolites (3). Autoregulatory molecules play a key role in controlling both the onset of cellular differentiation and secondary metabolism. The best studied autoregulator is A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), a small microbial hormone-like molecule (243 Da) that induces both morphological and physiological differentiation in Streptomyces griseus (4,5). The γ-butyrolactone regulatory system is widespread in Streptomycetes. Virginiae butanolids control virginiamycin production in Streptomyces virginiae (6) and SCB1 plays an important role in the control of actinorhodin and undecylprodigiosin biosynhesis and a cryptic, type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor (7,8). In Streptomyces griseus binding of Afactor to its cellular receptor ArpA derepresses expression of the transcriptional activator AdpA. While initially identified as the activator of streptomycin production through strR, this protein acts as a central switch, and the AdpA regulon includes several important positive regulators of development (ssgA, amfR, and adsA (bldN)) and secreted proteases, reviewed in (9, 10). A-factor deficient mutants are neither able to sporulate nor to produce antibiotics (streptomycin).
Another interesting autoregulator is the secreted signaling protein Factor C (Mw 34.555 KDa), originally isolated from the culture fluid of "Streptomyces griseus 45H" (11) which was recently shown to be identical to a laboratory strain known as Streptomyces flavofungini, itself a member of the Streptomyces albidoflavus species group (12). The Factor C producer strain like S. griseus readily sporulates in submerged culture (13). Similarly to Afactor, Factor C also plays a key role in cellular communication and cytodifferentiation.
A-factor mutants fail to develop aerial hyphae and spores and are therefore classified as bald mutants. Expression of facC from a low-copy plasmid in a spontaneous A-factordeficient bald mutant of S. griseus NRRL B-2682 restored its A-factor production as well as aerial mycelium and spore formation on solid media (14). The wild-type strain itself does not produce Factor C, as shown by immunoblotting (15) and by DNA hybridization studies (16).
Our previous results (14) indicate a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks. In preliminary experiments we observed characteristic differences between the extracellular proteomes of the strains that prompted detailed further analysis, facilitated by the currently available DNA sequence of the genome of S. griseus IFO13350 (17). Here we showed that the bald AFN mutant overexpressed several ABC transporter solute-binding proteins and stress response proteins compared to the wild type S. griseus B2682 strain or to the facC transformant of the AFN mutant in an effort to supply the cells with nutrients.

Strains and preparation of extracellular protein fractions
Strains of Streptomyces griseus were grown on R2YE agar plates (18) covered with a PCTE membrane (Poretics 0.2 μm pore size). The strains were S. griseus NRRL B-2682 (parental strain; in short B2682), its A-factor non-producing bald mutant S. griseus NRRL B-2682 AFN (AFN) and a transformant of AFN (designated AFN/pSGF4) that harbors facC on the pHJL401-based low-copy-number plasmid pSGF4 (16). Protein extracts were prepared from spent agar of surface-grown cultures by crumbling the solid medium and passing it through a syringe with frits at 4 o C by centrifugation. Samples of approximately 300 μg protein (measured using the Coomassie Protein Assay Reagent, Pierce) were purified using the ReadyPrep TM 2-D Cleanup Kit (Bio-Rad) according to the instructor's manual, and dissolved in Rehydration Buffer (8M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.2% 100x Bio-Lyte 3/10 (or 4/7) ampholyte, 0.002% Bromophenol Blue).

2D gel electrophoresis and image analysis
Separation of protein extracts (approximately 300 μg) in the first dimension was performed by isoelectric focusing using 17 cm long Immobiline DryStripsGels (IPG) in the pH range of 3-10 or 4-7 (Bio-Rad) on a Protean IEF cell (Bio-Rad). Samples were focused at 250 V for 15 minutes followed by an increase to 8000 V over 2.5 h and kept at this voltage for 45000 V-h.
Focused strips were separated on the basis of relative molecular weight in the second dimension on 13% SDS-polyacrylamide gels in a Protean II XL vertical gel system (Bio-Rad). For quantitative comparison of extracellular protein profiles gels were stained with colloidal Coomassie G-250 (19). Gels were scanned using a GS-800 Imaging Densitometer (Bio-Rad) and images were analyzed with PDQuest TM software (Bio-Rad). Histograms comparing spot quantity were generated with this software. Two-fold differences between the mutant and the parental B2682 were considered as significant changes. The data below are from a single representative experiment but at least two additional, biological replicas were performed and they showed similar results.

In-gel digestion
Gel slices containing 2D PAGE-separated proteins were cut and diced and then washed with Protein scores greater than 80 (full NCBI database search) or 51 (S. griseus database) were considered significant (P < 0.05).
Search parameters were as follows: mass accuracy: 200 ppm; only tryptic cleavages were permitted; and 2 missed cleavages were considered. Carbamidomethylation of Cys-residues was a fixed modification; methionine oxidation, protein N-acetylation and pyroglutamic acid formation from N-terminal Gln residues (and Glu), and Me esterification of Asp and Glu residues (CBB-staining side reaction proven by PSD (20) were the considered variable modifications. Esterified peptides were only accepted when the peptide was also detected without the modification. Protein identification was confirmed by sequence information  Table 1., except for protein ID#14 which is not present in the sequenced S. griseus IFO 13350 strain.

RNA isolation and RT-PCR
For transcript analysis, RNA was isolated from surface-grown cultures of S. griseus B2682 or its A-factor nonproducing derivative AFN. Mycelium was grown on R2YE agar plates and harvested after 36 hrs (onset of aerial growth) or 54 hrs (sporulation). RNA isolation and semi-quantitative RT-PCR was carried out twice, as described previously (21). 200 ng of RNA was used for each reaction (concentration was assessed using a Nanodrop® spectrophotometer). Oligonucleotides used for RT-PCR are presented in Supplementary material Table S1.

Extracellular proteome analysis
The extracellular proteomes of S. griseus B2682, of its A-factor non-producing spontaneous mutant (AFN) and of AFN complemented with a plasmid expressing Factor C (called AFN/pSGF4; (16)) were studied. AFN has a nonsporulating (bald) phenotype, and introduction of Factor C-expressing plasmid pSGF4 restores wild-type levels of sporulation to this strain (16). The choice for the extracellular proteome is a logical consequence of our focus on extracellular signaling mechanisms, with many of the proteins that are part of the Afactor/AdpA response regulon extracellularly localized.
To obtain an initial assessment of the protein expression profiles in the different strains, SDS-PAGE was performed, which revealed that several protein bands varied strongly in intensity. Considering the low separative capacity of 1D-gels the samples were analyzed further by 2D-proteome analysis, and initial analysis with a pI range of 3-10 revealed that the vast majority of the proteins appeared in the pI range of 4-7, which was then used for all experiments. Over 200 detectable protein spots were identified on each gel by colloidal Coomassie staining ( Figure 1). In total 42 spots differed significantly between the parent and the mutants and these were analyzed by MALDI-TOF mass spectrometry. In a previous study (14) we showed that Factor C acts by restoring A-factor production and normal sporulation to S. griseus AFN. Restoration of A-factor production also resulted an increase in the expression of several A-factor responsive secreted proteases to wild-type levels. However, at the time detailed analysis of the proteomes of AFN and AFN/pSGF4 was severely hampered by the lack of adequate genome information of S. griseus. Though our data now indicate (see below) that most proteins studied are highly conserved in S. coelicolor and S. avermitilis a single amino acid substitution in the peptides will prevent protein identification. This significant hurdle was overcome once the full genome sequence of S. griseus had become publicly available (17).
The main question we sought to address this time is what proteins are expressed or overexpressed in the AFN mutant to compensate for the lack of extracellular hydrolases that normally supply the developing colonies with nutrients. 16 out of 42 spots fulfilled the selection criteria (for details see the Materials and Methods section) and they were unambiguously identified ( Table 1 Table 1. Interestingly, all proteins with the exception of SodA are well conserved in S. avermitilis and S. coelicolor (Table 1) notably of glycine, serine, threonine and vitamin B6. SGR1498 is an ABC transporter solute binding protein that is known to be involved in xylose uptake (25). SGR2237 is a predicted arginine/ornithin binding protein and likely involved in the transport of polar amino acids.
SGR5275 is MalE or maltose-binding protein which is the solute binding protein belonging to the maltose uptake system in streptomycetes (26,27). SGR1737 is a glutamate ABC transporter substrate-binding protein and likely involved in sensing and processing of environmental information. SGR1498, SGR1737, SGR2237 and SGR5275 all have an amino-terminal secretion signal sequence and a conserved prokaryotic membrane lipoprotein lipid attachment site with a cystein residue that is most likely a site for ADP-ribosylation.
Finally, SGR5704 is an orthologue of the well-studied sporulation control protein Spo0M of Bacillus subtilis and most likely in an operon with the phospholipid binding protein SGR5703. Mutation/deletion in spo0M or overexpression of spoOM in B. subtilis results in impaired sporulation (28).
The identified proteins are all overexpressed in the AFN mutant, which is deficient in Afactor production. A-factor as a pleiotropic autoregulator in S. griseus during normal development induces the biosynthesis of several secreted hydrolases whose function is to digest biopolymers and supply the differentiating colony with nutrients (29,30,31). In the AFN mutant these hydrolases are not secreted (14), therefore the supply of small molecule nutrients is probably scarce.
Many of the proteins overexpressed in AFN are specific ligand-binding proteins associated to the transmembrane translocator component of the ABC transporters (31). It is likely that the overproduction of these nutrient scavenging proteins is a response to try and compensate for the reduced availability of nutrients. All of the ABC transporter substrate binding proteins have an amino-terminal secretion signal sequence and a properly positioned conserved prokaryotic membrane lipoprotein lipid attachment site with a cysteine residue.
Secretion of these proteins is regulated by ADP-ribosylation. When the cystein is ADPribosylated these proteins cannot be exported to the surface and are therefore nonfunctional in transport (32). ADP-ribosylation-mediated inactivation of substrate binding proteins most likely takes place particularly during normal growth and development, when the supply of nutrients is plenty.

Transcriptional analysis of the overexpressed proteins
To our surprise, many of the overexpressed proteins detected extracellularly are predicted lipoproteins whose final destination is the cell surface, and their amount in the secretome therefore does not necessarily reflects their gene expression. To analyze if the enhanced accumulation of proteins in the AFN mutant was at least in part due to enhanced transcription, RNA was isolated from surface-grown cultures of S. griseus B2682 and its AFN mutant derivative. For this, mycelium was grown on R2YE agar plates and harvested at the time when aerial mycelium was formed as well as during sporulation. We used rpsI (SGR2801, encoding ribosomal protein S9) as positive control, and reactions without reversed transcriptase step were performed to confirm the lack of DNA contamination in the samples. As shown in Figure 3, while rpsI transcript levels were the same in wild-type and mutant and did not vary in time, transcription of SGR1460, 1498, 1737, 2237, 2245, 3109, 5275, 5280 and 5704 was strongly upregulated in the AFN mutant during early aerial growth.
This corresponds to the critical phase in the Streptomyces life-cycle when programmed cell death is initiated to provide the cells with nutrients. The transcriptional analysis shows that the enhanced protein levels observed in the proteome of AFN (Figures 1-2) is at least in part due to enhanced gene expression in the absence of A-factor. Interestingly, differences in transcript levels were far less pronounced during sporulation, with only SRG1460 and SGR3109 upregulated at this time point, while SGR5704 was lower in the mutant than in the parent. This strongly suggests that transcriptional upregulation is specific for the time corresponding to programmed cell death.
Interestingly, we also observe elevated protein levels for the stress-related proteins in the AFN mutant during sporulation, which is not explained by differences at the transcriptional level alone. A likely explanation is provided by the fact that A-factor activates a multitude of extracellular proteases (10, 14), and hence secreted proteins are expected to be much more stable in the A-factor nonproducing mutant AFN, further contributing to the accumulation of proteins in the mutant

ABC transporters and the control of Streptomyces development
Streptomycetes are soil-dwelling bacteria that are able to utilize almost all naturally occurring polymers, such as starch, cellulose, chitin and agar. These polymers are degraded by extracellular hydrolases and subsequently imported (mainly as monomers or dimers) via specific permeases, either via ABC transporters or via the phosphotransferase system PTS (34, 35, 36, 37). Permeases that are involved in sensing and transport therefore substantially influence their metabolism and morphogenesis. An in silico survey (25) identified 81 ABC transporters in S. coelicolor that are involved in the uptake of sugars, oligopeptides and other nutrients. This large number of transporters and secreted hydrolases perfectly reflects the lifestyle of these microbes. In a recent analysis of the membrane-associated proteome (38) in S. coelicolor it was noted that a large proportion of these characterized proteins related to ABC transporter systems, including SCO5776, SCO2231 and SCO6009 whose orthologues we found in this study, which was considered as a snapshot of the nutritional requirement of the organism.
Interestingly, recent studies revealed that in fact several sugar transporters play a crucial role in the control of development, and in particular those that relate to Nacetylglucosamine transport and metabolism. These include the universal PTS components EI, EIIA Crr and HPr (36, 2) as well as components of the DasABC transporter. DasABC transports chitobiose, the dimer of N-acetylglucosamine, and DasA is a lipoprotein that functions as the chitobiose binding protein (39). Besides in the transport of chitobiose DasA also plays a role in nutrient sensing, perhaps by interacting with the chiRS two-component system (34). The dasABC operon is developmentally regulated and plays a role in the control of sporulation in an A-factor independent manner in S. griseus (40,41), and deletion of dasA results in a non-sporulating (bald) phenotype in S. coelicolor (34). Other oligomeric transporters that relate to the onset of development are for example the BldK oligopeptide transporter of S. coelicolor that is essential for sporulation on rich and glucose-containing media (42) and a second developmentally regulated ABC transporter -SCO7167 -involved in morphogenesis of S. coelicolor (43).

Superoxide dismutases in Streptomyces
Superoxide dismutases are a ubiquitous part of the cellular defense system against oxidative stress. They convert the toxic superoxide to hydrogen peroxide which is further degraded by the enzyme catalase. So far in Streptomyces two classes of superoxide dismutases have been described, namely the nickel containing SodN and the iron-zinc containing SodF. We detected an additional superoxide dismutase, which has high similarity to the Bacillus pumilus SAFR-032 manganese superoxide dismutase SodA (YP_001487459). This protein so far has not been described in Streptomyces and may therefore represent a third class. In S. pristinaespiralis the expression of SodF is regulated by the SpbR protein, an analogue of the S. griseus ArpA and thus relating to the γ-butyrolactone regulon (44). It is also hypothesized that SodF is involved in adaptation to oxidative stress generated by metabolic shifts during colony development (44). In further support of this hypothesis, a catalase is also developmentally regulated and required for aerial mycelium formation in S. coelicolor (45).
Previously (14) we identified SodF in S. griseus as upregulated in mutant strain AFN relative to the parental strain S. griseus B2682, suggesting that SodF is negatively controlled by AdpA. In this work we identified SodN as a protein upregulated in the AFN mutant.
Interestingly, the presence of nickel in a micromolar concentration in the growth media induced the expression of SodN in S. coelicolor, whereas the expression of SodF was repressed (46). The differential expression of superoxide dismutases seem to suggest that a strict control of the level of SOD is of great importance to cellular defenses against oxidative stress.
The enhanced expression of the nutrient and environmental stress proteins in a strain that fails to produce (significant amounts of) A-factor underlines the stress that is generated by the absence of this signaling molecule.
The A-factor nonproducing strain of S. griseus detailed in this work is a valuable asset in the search for genes that control the production of A-factor and the response it elicits. The afn locus does not correspond to any of the known A-factor biosynthetic genes or regulatory genes, such as afsA, adpA, or arpA. Considering the fact that the afn mutant is able to produce A-factor under some conditions (such as after addition of the signaling protein Factor C) we anticipate that the mutation lies in a gene involved in the regulation of A-factor biosynthesis. The nature of the afn mutation and its role in the control of A-factor production is currently under investigation in our laboratories.   Table 1.