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Molecular & Cellular Proteomics 6:1248-1256, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

The Secreted Signaling Protein Factor C Triggers the A-factor Response Regulon in Streptomyces griseus

Overlapping Signaling Routes^*,S

Zsuzsanna Birkó, Sylwia Bialek, Krisztina Buzás^¶,||, Emília Szájli^¶, Bjørn A. Traag, Katalin F. Medzihradszky^¶,**, Sebastien Rigali, Erik Vijgenboom, András Penyige, Zoltán Kele, Gilles P. van Wezel^, and Sándor Biró^,¶¶

From the Department of Human Genetics, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei körút 98, H-4032 Debrecen, Hungary, Microbial Development, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands, ^¶ Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Temesvári körút 62, H-6726 Szeged, Hungary, ^|| Kromat Kft, Sirály utca 3, H-1124 Budapest, Hungary, ^** Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, and Department of Medicinal Chemistry, Faculty of Medicine, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION Note Added in Proof-- REFERENCES

 
Members of the prokaryotic genus Streptomyces produce over 60% of all known antibiotics and a wide range of industrial enzymes. A leading theme in microbiology is which signals are received and transmitted by these organisms to trigger the onset of morphological differentiation and antibiotic production. The small -butyrolactone A-factor is an important autoregulatory signaling molecule in streptomycetes, and A-factor mutants are blocked in development and antibiotic production. In this study we showed that heterologous expression of the 324-amino acid secreted regulatory protein Factor C resulted in restoration of development and enhanced antibiotic production of an A-factor-deficient bald mutant of Streptomyces griseus, although the parental strain lacks an facC gene. Proteome analysis showed that in the facC transformant the production of several secreted proteins that belong to the A-factor regulon was restored. HPLC-MS/MS analysis indicated that this was due to restoration of A-factor production to wild-type levels in the transformant. This indicates a connection between two highly divergent types of signaling molecules and possible interplay between their regulatory networks.

Another interesting autoregulator is the secreted signaling protein Factor C (molecular mass, 34.555 Da), originally isolated from the culture fluid of "S. griseus 45H" but recently shown to be identical to a laboratory strain known as Streptomyces flavofungini, itself a member of the Streptomyces albidoflavus species group.¹ The Factor C producer strain, like S. griseus, readily sporulates in submerged culture (9, 10). Similarly to A-factor, Factor C also plays a key role in cellular communication and cytodifferentiation. Expression of facC from a low copy plasmid in a spontaneous A-factor-deficient bald mutant of S. griseus NRRL B-2682 restored its aerial mycelium and spore formation on solid medium. This strain does not produce Factor C as shown by immunoblotting (11) and by DNA hybridization studies (12). This suggested that the Factor C response regulon acts independently or has a complex interaction with the A-factor regulon. In preliminary experiments comparing one-dimensional protein gels of the above strains we observed characteristic differences between the extracellular proteomes of the strains that prompted us to perform a detailed analysis. Here we compare the extracellular proteomes of S. griseus NRRL B-2682 and its A-factor-non-producing bald mutant with the same strain expressing Factor C from a low copy number vector.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION Note Added in Proof-- REFERENCES

 
Strains and Preparation of Extracellular Protein Fractions—
Strains of S. griseus were grown on R2YE agar plates (13) covered with a polycarbonate track-etched 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 (in short AFN), and a transformant of AFN (designated AFN/pSGF4) that harbors facC on the pHJL401-based low copy number plasmid pSGF4 (14). 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 °C by centrifugation. Samples of 300 µg of protein (measured using the Coomassie protein assay reagent, Pierce) were purified using the ReadyPrep^TM 2D² cleanup kit (Bio-Rad) according to the instruction manual and dissolved in Rehydration Buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% 100x Bio-Lyte 3/10 (or 4/7) ampholyte, 0.002% bromphenol blue).

2D Gel Electrophoresis and Image Analysis—
Separation of protein extracts (300 µg) in the first dimension was performed by isoelectric focusing using 17-cm-long Immobiline DryStrip gels (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 min followed by an increase to 8000 V over 2.5 h and kept at this voltage for 45,000 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 (15). 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. 2-fold differences compared with 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, diced, and then washed with 25 mM NH₄HCO₃ in 50% (v/v) acetonitrile/water. After reduction with 10 mM DTT (30 min at 56 °C) and alkylation with 55 mM iodoacetamide (30 min at room temperature in the dark) the proteins were digested with side chain-protected porcine trypsin (Promega, Madison, WI) at 37 °C for 4 h. Tryptic digests were extracted and desalted on C₁₈ ZipTips (Millipore, Bedford, MA). Mass spectrometric analysis of the unfractionated tryptic digests was performed in positive ion, reflectron mode on a Reflex III MALDI-TOF mass spectrometer (Bruker, Karlsruhe, Germany) using 2,5-dihydroxybenzoic acid as the matrix. Two-point external calibration was applied; this guarantees a mass accuracy within 200 ppm. The peak lists were generated with flexAnalysis (version 2.0) software: peak detection algorithm, Sophisticated Numerical Annotation Procedure; signal-to-noise threshold, 5; quality factor threshold, 30; sodium adducts; and trypsin autolysis products were deleted from the lists. Masses detected were submitted to a database search with MS-Fit in ProteinProspector version 4.23.4 in the National Center for Biotechnology Information non-redundant (NCBInr) database (July 18, 2006; 3,794,285 sequences).

Search parameters were as follows: mass accuracy, 200 ppm; only tryptic cleavages were permitted; and two 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 Me esterification of Asp and Glu residues (Coomassie Brilliant Blue-staining side reaction (16) proven by PSD) 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 obtained from MS/MS (postsource decay; data shown in Supplemental Figs. S1–S9) spectra acquired in 10–12 steps, lowering the reflector voltage by 25% in each step, and then stitching the data together. Some MS/MS experiments were performed on an Agilent XCT plus ion trap equipped with an atmospheric pressure MALDI source using 4-OH--CN-cinnamic acid as the matrix. Search parameters for MS/MS data were 200 ppm for the precursor ion and 1 Da for the fragment ions. Cleavage specificity and covalent modifications were considered as described above. Instead of the accession numbers the locus is listed in our tables thus eliminating the redundancy originated from multiple entries for the same amino acid sequence. When there were multiple entries corresponding to slightly different sequences only the database entry with the most matching peptides was included.

Measurement of Antibiotic Concentration—
Antibiotic concentration in the supernatants of cultures grown in soybean medium (2% corn-steep liquor, 2% soybean meal, 0.3% NaCl, 0.2% CaCO₃, 0.05% MgSO₄·7H₂O, 2% glucose, pH 7.5) was determined by an agar diffusion method. Antibiotic medium (0.5% meat extract, 0.1% Na₂HPO₄, 0.01% KH₂PO₄, 0.05% peptone) was mixed with Bacillus subtilis ATCC 6633 spores. In brief, wells were cut in the agar and filled with either 100 µl of the concentrated samples, of 10-fold diluted samples, or of a streptomycin standard dilution series and were incubated at 37 °C for 18 h. Diameters of the cleared zones were linear over the range studied (0–10 µg ml^–1).

Prediction of Adp Binding Sites—
Multiple alignments and position weight matrices were generated with the Target Explorer automated tool (17, 18). The weight matrix for AdpA was generated from a set of experimentally validated AdpA binding sites (19).

Extraction of A-factor from Liquid-grown Cultures—
To assay the production of A-factor, the strains were grown in GYM (glucose, yeast extract, malt extract) medium (20) in submerged cultures in a rotary shaker at 230 rpm at 28 °C for 72 h. The complete culture (400 ml) was extracted with an equal amount of chloroform, and the organic phase was evaporated in a vacuum (Rotadest, Buchi; 60 °C). The concentrated extract was then redissolved in 200 µl of ethanol.

Biological Assay of A-factor—
The presence of A-factor in extracts was determined using a modified version of an agar diffusion assay (21). For this, the test strains were streaked on GYM or DM1-agar medium (20), a paper disc containing 20 µl of extract or authentic A-factor (1 mg/ml in EtOH; Funakoshi Co. Ltd. Code Number KA106) was placed on the agar plates close to the bacteria, and plates were incubated at 28 °C for 96 h. As indicator strains we used S. griseus HH1 (22) or S. griseus AFN. Restoration of aerial mycelium and spore production were inspected.

Determination of A-factor by Mass Spectrometry—
MS measurements were obtained on a Finnigan TSQ 7000 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a Finnigan atmospheric pressure chemical ionization source. The instrument was operated in positive ion mode using selective reaction monitoring and fragment ion scan mode. In selective reaction monitoring mode the first quadrupole was set to select m/z 243 (M + H⁺ of A-factor), which was fragmented in the collision cell, and the three most intense fragments (m/z 127, 109, and 67) were measured with the last quadrupole. For quantitative determination of A-factor the most intense mass chromatogram (m/z 109) was used. The atmospheric pressure chemical ionization needle was adjusted to 4 µA, and N₂ was used as a nebulizer gas. The collision potential was 20 eV. Argon was used as the collision gas, and the pressure in the collision cell region was 2 millitorrs.

Samples were subjected to short gradient HPLC separation before MS analysis. Mobile phases were: solvent A, water and formic acid (99.95:0.05, v/v); solvent B, methanol, water, and formic acid (80:19.95:0.05, v/v/v). The HPLC apparatus was Applied Biosystems 140C. Conditions were as follows: flow rate, 250 µl min^–1; gradient, 0–100% B in 10 min; column, home-packed Nucleosil C₁₈, 20-µm-diameter x 2.0-mm length.

RNA Isolation—
Mycelium was harvested from liquid-grown cultures of S. griseus B2682 and its A-factor-non-producing mutant AFN. Strains were grown in TSBS (tryptone, soya broth, sucrose medium) until an A₅₅₀ of 0.6, washed in MM without carbon source, and transferred to MM with 0.5% (w/v) mannitol as the carbon source and 0.1% casamino acids. This elicits production of A-factor among others leading to submerged sporulation. Cultures were incubated at 30 °C, and RNA was isolated after 0, 20, 60, and 180 min. RNA was purified using the Kirby-mix protocol (13). RNA purification columns (RNeasy, Qiagen) and DNase I treatment were used as well as salt precipitation (final concentration, 3 M sodium acetate, pH 4.8) to purify the RNA and fully remove any traces of DNA, respectively. Before use, the RNA preparations were checked for their quality and integrity on an Agilent 2100 Bioanalyzer (Agilent Technologies).

PCR Amplification and Sequencing of afsA in S. griseus AFN—
PCRs were performed with Pfu polymerase (Stratagene) in the presence of 10% (v/v) DMSO. The program used was as follows: 2 min at 95 °C followed by 35 cycles of 1 min at 95 °C, 1 min at 58 °C, and 2 min at 72 °C. The primers were AfsA-For (5'-ctggaattccggtaaacggcgcggcctgtgag) and AfsA-Rev (5'-agtcagatctatccgcacgggtccggcatccgccag). DNA sequencing directly on the PCR-amplified DNA was performed by BaseClear BV (Leiden, The Netherlands).

Transcriptional Analysis of afsA by RT-PCR—
Transcriptional analysis of afsA was carried out by RT-PCR using the Superscript III one-step RT-PCR system with Platinum® Taq DNA polymerase (Invitrogen). For each RT-PCR 100 ng of RNA was used together with 1 µM final concentration of each primer. The program used was as follows: 45-min cDNA synthesis at 48 °C followed by 2 min at 95 °C and 35 cycles of 45 s at 94 °C, 30 s at 68 °C, and 30 s at 68 °C. The reaction was completed by 5-min incubation at 68 °C. 5 µl of each sample was visualized by ethidium bromide staining after electrophoresis on a 2% agarose gel in 1x Tris acetate-EDTA buffer. The oligonucleotides were afsA_RTfor (5'-gaagcggtccttccgcacgacca) and afsA_RTrev (5'-gcccgccacttcaggtcggagca). Quantification of the RT-PCRs was done by scanning the gels using the GS-800 imaging densitometer and analysis using Quantity One software (Bio-Rad). For control experiments, to rule out DNA contamination of the RNA samples, the same experiments were repeated under identical conditions but after inactivation of the reverse transcriptase by incubation for 5 min at 95 °C.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION Note Added in Proof-- REFERENCES

 
Extracellular Proteome Analysis—
To study the effect of Factor C on protein expression, we analyzed the extracellular proteomes of S. griseus B2682, of its A-factor-non-producing spontaneous mutant (AFN) that had a bald phenotype, and of the AFN strain with restored sporulation due to complementation with a plasmid expressing Factor C (called AFN/pSGF4 (14)). Our choice for the extracellular proteome relates to our focus on signaling and to the fact that many of the AdpA target proteins are extracellularly localized.

To obtain an initial assessment of the protein expression profiles in the different strains, SDS-PAGE was performed, revealing that indeed several protein bands varied strongly in intensity. Considering the low separative resolution of one-dimensional gels the samples were analyzed further by 2D electrophoresis. The vast majority of proteins in the samples collected after 3 and 4 days of growth on R5 solid cultures appeared in the pH range 4–7 when an IPG strip of pH 3–10 was applied. Further separations, using the narrower isoelectric point range pH 4–7 in the first dimension and a molecular mass range between 6.5 and 200 kDa in the second dimension, produced 200–240 detectable spots on each gel after colloidal Coomassie staining (Fig. 1). We focused on proteins whose expression differed at least 2-fold between the studied strains. 50 spots that fulfilled this criterion were isolated and analyzed by MALDI-TOF mass spectrometry. The main question we sought to address was: what is the response triggered by Factor C that results in the restoration of development to the AFN strain? Although at the time of this study two complete Streptomyces genome sequences (S. coelicolor and Streptomyces avermitilis) and a nearly complete one (Streptomyces scabies) were available, the lack of a complete S. griseus database severely hampered our efforts in the identification of the relevant proteins. Nine of 50 spots were unambiguously identified (Tables I and II).

View larger version (60K): [in this window] [in a new window]   FIG. 1. 2D gel electrophoresis maps of secreted proteins extracted from 72-h-old surface-grown cultures of B2682 (a), AFN (b), and AFN/pSGF4 (c) and from 96-h-old surface-grown cultures of B2682 (d), AFN (e), and AFN/pSGF4 (f). Numbers correspond to the ID numbers of proteins in the tables.

View larger version (47K): [in this window] [in a new window]   FIG. 2. Quantitative image analysis of the proteins performed by PDQuest 2D analysis software (Bio-Rad) in 72-h-old cultures (A) and 96-h-old cultures (B). Numbers underneath each plot correspond to the protein ID numbers in Fig. 1 and Tables I, II, and III. The bars represent the relative amount of the different proteins in the strains in the order of B2682 (a), AFN (b), and AFN/pSGF4 (c). SSP, standard spot number; INT, intensity.

Several of the proteins whose expression is restored approximately to the original parental levels by Factor C, namely SprU, SGPA, SgmA, and StrU, have been identified previously by Horinouchi and co-workers (8) as members of the AdpA regulon in S. griseus. It was also reported that their expression is activated in a growth phase-dependent manner. Cpase SG (24) is also a possible development-related protein as it has significant similarity to the sporulation-related zinc peptidase I in Bacillus sphaericus (34). Of the known AdpA-dependent genes, sprA, sgmA, and sprU are all strongly transcribed after 72 h (23, 26, 27). We could readily identify all gene products in samples prepared from 3–4-day-old mycelia, suggesting possible function in the control of development. The substrate mycelium is lysed during aerial mycelium formation, and a likely function for hydrolytic enzymes such as proteases, nucleases, and lipases is to degrade the cytoplasmic contents and supply material to the newly forming aerial mycelium (35).

Restoration of the expression of StrU by facC is particularly interesting as this shows that not only development is restored by Factor C to an A-factor-non-producing bld mutant but perhaps also antibiotic production. AdpA transmits the upstream A-factor signal directly to the streptomycin biosynthesis genes via the pathway-specific activator strR (28, 36–38). To assess whether indeed streptomycin production was enhanced by Factor C we measured antibiotic production of B2682, AFN, and AFN/pSGF4. Although we failed to detect significant amounts of streptomycin in either B2682 or AFN (i.e. below the detection limit of about 1 µg ml^–1 for the agar diffusion method used), significant amounts of streptomycin were detected in the spent culture fluid of AFN/pSGF4 with 8.2 µg ml^–1 after 48 h and 11 µg ml^–1 after 72 h of growth in liquid culture. This strong increase in antibiotic production corresponds well to the observed enhanced expression of StrU relative to that in AFN or B2682.

To further characterize the other targets, we subjected all genes corresponding to the identified proteins (Table I) to an extensive in silico analysis using the Target Explorer program (18) with all known AdpA binding sites as input for the production of a reliability matrix. Our predictions (Table III) suggest that all may be controlled by AdpA with statistically reliable sites upstream of scpD, sgaP, pstS, and sodF. All of these had not yet been identified as members of the AdpA regulon.

Importantly the majority of the targets are metal-binding proteins, most of which use zinc ions as cofactor, and carry the twin arginine translocation pathway signature for Tat secreted proteins (Table II). This pathway is responsible for the export of folded proteins together with their cofactors across the cytoplasmic membrane (39). Factor C itself binds efficiently to a zinc column and has a zinc binding site that shows similarity to that of eukaryotic zinc finger proteins, including zinc finger proteins 275 and 502 of different animal species (40). Like most of the targets discussed in Table I, Factor C also has a secretion signal that conforms to the consensus signal sequence of the twin arginine translocation pathway and is therefore predicted to be exported via the Tat secretion pathway.

A-factor Production of the Strains—
Restoration of development of AFN by Factor C and the observed high similarity between the extracellular proteomes of AFN/pSGF4 and the wild-type strain B2682 prompted the analysis of A-factor production by B2682, AFN, AFN/pSGF4, and S. griseus 45H. Two independent methods were used: a biological assay (21) and mass spectrometry. In the biological assay we used S. griseus HH1 (22) and AFN as test strains. Authentic A-factor and extracts prepared from B2682 and AFN/pSGF4 restored aerial mycelium formation of the S. griseus HH1 strain, whereas those prepared from AFN (Supplemental Fig. S10) or S. griseus 45H (not shown) had no effect. Aerial mycelium and spore formation were also restored by authentic A-factor and by B2682 and AFN/pSGF4 extracts but not by S. griseus 45H extracts when AFN was used as test strain (Supplemental Fig. S11). This strongly suggested that expression of facC restored A-factor production to the AFN strain.

This observation was further substantiated by the HPLC-MS/MS analysis of the cell extracts by comparison of the MS/MS of the A-factor standard and the cell extracts prepared from B2682, AFN/pSGF4, and AFN (Fig. 3). The mass of the captured fragments was identical in all four cases and in good agreement with the results published recently (41). Quantitative assessment of A-factor production of the different strains based on the measurement of the intensity of the most characteristic peak (109.2 Da) showed that A-factor is produced by B2682 (2.3 mg ml^–1) and by AFN/pSGF4 (2.0 mg ml^–1) strains in nearly equal amounts. In contrast, only trace amounts (0.0012 mg ml^–1, which is 1500–2000 times less than in B2682 and AFN/pSGF4 or in other words pg ml^–1 in the original culture) of A-factor were produced by AFN, and none was produced by S. griseus 45H (Supplemental Table S1 and Figs. S12–S15). Although AFN produces only a very small peak corresponding to A-factor, it is most likely A-factor as the fragmentation pattern is identical, producing the same characteristic peaks of 127, 109, and 67 Da. This implies that the A-factor biosynthetic genes are still functional in AFN and that instead the activation of A-factor biosynthesis has been lost. This makes AFN a very important mutant as uncovering the nature of this mutation would better our understanding of the control of A-factor biosynthesis.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Comparison of MS/MS of authentic A-factor (A), B2682 (B), AFN/pSGF4 (C), and AFN (D) extracts.

To establish whether afsA transcription was affected in S. griseus AFN, RNA was isolated from both strains and analyzed by semiquantitative RT-PCR. A-factor production and the subsequent submerged sporulation by S. griseus are triggered by nutritional shift-down, and these conditions were therefore chosen to ascertain significant transcription of afsA. Under the conditions chosen, small clublike conidia were formed by the parental strain B2682 at the tips of the hyphae after 1.5–2 h following nutritional shift-down, whereas submerged sporulation started some 5 h later. Expectedly bld mutant AFN did not produce sporogenic submerged hyphae. The RNA was analyzed by semiquantitative RT-PCR using oligonucleotides afsA_RTfor and afsA_RTrev. afsA transcription was only slightly induced by the nutritional shift-down; interestingly total transcript levels were significantly higher in the mutant than in the parent (Fig. 4). In a control experiment where reaction conditions and enzymes were identical except that the reverse transcriptase was heat-inactivated, no products were obtained, confirming the absence of DNA contamination. Hence S. griseus AFN has a wild-type and actively transcribed afsA gene. Considering that AFN is probably able to produce A-factor, this strongly suggests that the defect in AFN lies in another yet unknown gene involved in the control of A-factor biosynthesis. The overexpression of the A-factor biosynthetic gene afsA in AFN mutants is best explained as an (unsuccessful) attempt to compensate for the absence of A-factor. Recently we observed a similar effect when we analyzed S. coelicolor mutants deficient in the cell division activator gene ssgA.³ SsgA is involved in the localization of septa, and the cell machinery itself is unaffected in ssgA mutants; the lack of septal peptidoglycan synthesis prompted strong up-regulation of ftsI, which encodes the penicillin-binding protein responsible for synthesis of the septal peptidoglycan. Hence in both cases the genes (afsA or ftsI) normally responsible for the missing compounds (A-factor and septal peptidoglycan, respectively) are up-regulated. Whether this interesting concept is true for other vital biosynthetic pathways needs further investigation.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Analysis of the transcription of afsA. Semiquantitative RT-PCR was performed on RNA samples isolated from S. griseus strains B2682 and its spontaneous bald mutant AFN 0, 20, 60, and 180 min after nutritional shift-down (from rich (TSBS) to MM liquid cultures). Control experiments on 16 S rRNA and without reverse transcriptase showed the integrity of the RNA and the lack of DNA, respectively, in the samples (data not illustrated).

	Note Added in Proof—

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION Note Added in Proof-- REFERENCES

 
Recently new insights on A-factor biosynthesis in S. griseus were provided by the Horinouchi group (Kato, J., Funa, N., Watanabe, H., Ohnishi, Y., and Horinouchi, S. (2007) Proc. Nat. Acad. Sci. U.S.A. 104, 2378–2383.

	ACKNOWLEDGMENTS

 
We thank Anikó Szilágyi for the excellent technical support in A-factor extraction.

	FOOTNOTES

 
Received, September 22, 2006, and in revised form, March 19, 2007.

Published, MCP Papers in Press, March 20, 2007, DOI 10.1074/mcp.M600367-MCP200

¹ Z. Kiss, A. C. Ward, Z. Birkó, K. F. Chater, and S. Biró, unpublished data.

² The abbreviations used are: 2D, two-dimensional; Cpase, carboxypeptidase; SGAP, S. griseus leucine aminopeptidase; SGPA, Streptogrisin A precursor; Tat, twin arginine translocation; ID, identification; MM, minimal medium.

³ E. Noens, V. Mersinias, C. P. Smith, and G. P. van Wezel, unpublished data.

^* The work was supported in part by Netherlands Organisation for Scientific Research (NWO) Grant 048.011.049 and Hungarian Scientific Research Fund (OTKA) Grant N42824 in frame of the Hungarian-Dutch Research Cooperation 2002 (to G. P. v. W. and S. B.) and OTKA Grant T 034147 (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

To whom correspondence may be addressed. E-mail: g.wezel{at}chem.leidenuniv.nl

^¶¶ To whom correspondence may be addressed. E-mail: sbiro{at}dote.hu

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION Note Added in Proof-- REFERENCES

 

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