Identification of DNA-binding proteins involved in regulation of expression of the Streptomyces aureofaciens sigF gene, which encodes sporulation sigma factor {sigma}F

D. Homerová1, B. Sevcíková1, O. Sprusanský1 and J. Kormanec1

Institute of Molecular Biology, Slovak Academy of Sciences, Dubravská cesta 21, 842 51 Bratislava, Slovak Republic1

Author for correspondence: J. Kormanec. Tel: +421 7 5941 2432. Fax: +421 7 5477 2316. e-mail: umbijkor{at}savba.sk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of the sigF gene encoding a sporulation-specific sigma factor, {sigma}F, in Streptomyces aureofaciens is restricted only to sporulation. Gel mobility-shift assays using protein fractions from different developmental stages of S. aureofaciens revealed two different putative proteins specifically bound to the sigF promoter region: a protein (designated RsfA) present in young substrate mycelium, and a protein (designated RsfB) present in the course of sporulation. Based on the characteristic profiles of their appearance during differentiation, RsfA might be a repressor and RsfB an activator of sigF expression. The location of a specific binding site of the repressor-like protein (RsfA) was determined by gel mobility-shift assays of promoter deletion fragments and by DNase I footprinting analysis. The binding site mapped from nucleotides -87 to -25 relative to the transcription start point of the sigF promoter, and overlapped the -35 promoter region. Given the dependence of sigF expression upon whiH, the putative sporulation transcription factor WhiH was overproduced in Escherichia coli and used in the mobility-shift assays with the sigF promoter. However, no specific binding was detected, indicating an indirect dependence of sigF upon whiH.

Keywords: Streptomyces aureofaciens, differentiation, repressor, promoter, sigma factor

Abbreviations: tsp, transcription start point


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomycetes are Gram-positive soil bacteria undergoing an exceptional process of morphological differentiation, phenotypically similar to the sporulation of eukaryotic filamentous fungi. The process is regulated at several levels, and in spite of identification of several regulatory genes important in regulation of the process, the detailed mechanism of the regulation of differentiation in streptomycetes still remains obscure (Chater, 1998 ).

Developmental processes in higher as well as in lower organisms involve the sequential temporal and spatial expression of regulatory genes, which ensures strictly regulated expression of the genes necessary for the morphology and physiology of differentiated cells. The regulation of gene expression at the transcriptional level by heterogeneity of {sigma} factors of RNA polymerase belongs to the key mechanisms of regulation of differentiation in several bacteria. In the simplest, but the most studied model of bacterial differentiation, Bacillus subtilis, the differentiation, comprising endospore formation, is controlled by successive activation of a cascade of {sigma} factors (Kroos et al., 1999 ). Two genes encoding {sigma} factors having a function in regulation of sporulation have also been identified in Streptomyces. whiG of Streptomyces coelicolor (Chater et al., 1989 ) and its functional homologue rpoZ in Streptomyces aureofaciens (Kormanec et al., 1994 ) have a role in the initiation of differentiation of aerial hyphae into spore chains. The second gene, sigF, is essential in the late stage of differentiation, in spore maturation and pigmentation in both strains (Potúcková et al., 1995 ; Rezuchová et al., 1997 ).

Differentiation of S. aureofaciens has several features in common with that of the genetically best studied S. coelicolor strain. However, morphologically there are some differences between these two strains (Kormanec et al., 1998 ). Though several regulatory proteins are common, the regulation of differentiation has some specific features in each strain (Kormanec et al., 1994 , 1996 , 1998 , 1999 ). Expression of the sigF gene, encoding the late-sporulation-specific {sigma}F, is temporally committed to sporulation in both strains (Kelemen et al., 1996 ; Kormanec et al., 1996 ). Sun et al. (1999 ) have recently shown that expression of sigF is spatially limited to the spore compartment, with no evidence of its expression before sporulation septa are formed in S. coelicolor. The dependence of sigF expression upon early-sporulation-specific {sigma} factor gene whiG/rpoZ in both strains indicated a cascade of activation, similar to that occurring in B. subtilis (Kelemen et al., 1996 ; Kormanec et al., 1996 ). Moreover, besides whiG, sigF expression has also been shown to depend upon other five whi genes (whiA, B, H, I and J) that are required for sporulation septum formation in S. coelicolor (Kelemen et al., 1996 ). However, sigF dependence upon the {sigma} factor encoded by whiG/rpoZ is indirect, and the sigF promoter is probably recognized by a holoenzyme of RNA polymerase containing some other {sigma} factor (Kelemen et al., 1996 ; Kormanec et al., 1996 ). Since at least three of the whi genes (whiB, H and I) encode potential regulatory proteins (Aínsa et al., 1999 ; Davis & Chater, 1992 ; Ryding et al., 1998 ), it seems likely that sigF expression might be activated by some of these regulatory proteins to ensure its correct temporal and spatial location in the spore compartment.

In the present study, we have investigated proteins which specifically bind to the S. aureofaciens sigF promoter region in the course of differentiation. One such protein (RsfA), which was present only in young substrate mycelium, was investigated in more detail. Its binding site was determined by DNase I footprinting, and was found to overlap the sigF promoter region. Given that sigF expression takes place only during sporulation, it seems likely that RsfA represents a repressor-like protein having a role in inhibition of sigF expression in substrate mycelium. S. aureofaciens sporulation-specific putative transcription factor WhiH (Kormanec et al., 1999 ) was overproduced in Escherichia coli as a whole protein, and with 6xHis-tagged N-terminal fusion. However, the protein showed no binding to the sigF promoter.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
S. aureofaciens CCM 3239 wild-type was from the Czechoslovak Collection of Microorganisms, Brno, Czech Republic. E. coli XL1Blue (Stratagene) was used as a host, and plasmid pBluescript II SK(+) (Stratagene) was used for E. coli cloning experiments. E. coli expression plasmids pET28a and pET11c were from Novagen. The overexpression was done in E. coli BL21(DE3)pLysS (Novagen). For cell-free extract preparation, 108 spores of S. aureofaciens were inoculated into 20 ml liquid medium NMP (Hopwood et al., 1985 ) containing different carbon sources at 0·5% (w/v) final concentration, and cultured at 30 °C to early-exponential phase (14 h), late-exponential phase (20 h) and stationary phase (36 h). For surface cultures, 108 spores of S. aureofaciens were spread on sterile cellophane membranes placed on Bennet medium (Horinouchi et al., 1983 ), and grown for 13 (substrate mycelium), 19 (the beginning of aerial mycelium formation), 36 (aerial mycelium approximately at the time of septation) and 60 h (spore maturation). Conditions for E. coli growth and transformation were as described by Ausubel et al. (1987 ).

DNA manipulations.
DNA manipulations in E. coli were done as described in Ausubel et al. (1987 ), and those in Streptomyces according to Hopwood et al. (1985 ). DNA fragments were isolated from agarose gel by the GeneClean technique (BIO 101). Nucleotide sequencing was performed by the chemical method (Maxam & Gilbert, 1980 ). Site-directed mutagenesis was done by the Chameleon mutagenesis kit from Promega.

Preparation of cell-free extracts.
Liquid-grown S. aureofaciens mycelium was harvested by centrifugation at 12000 g for 10 min, and washed with ice-cold STE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 8). Solid-grown mycelium was scraped from the cellophane. All successive steps were carried out at 4 °C. The mycelium was disrupted by grinding with purified acid-washed sea sand in the presence of liquid nitrogen in a mortar for about 5 min. The mixture was then suspended in binding buffer (12·5 mM Tris pH 7·9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 12% glycerol), and cell debris was removed by centrifugation for 30 min at 30000 g. The cell-free extracts were stored in aliquots at -70 °C. The extracts were used in gel mobility-shift assays and DNase I footprinting experiments.

Partial purification of the DNA-binding protein RsfA.
S. aureofaciens was cultured in NMP medium with 0·5% (w/v) glucose at 30 °C for 14 h. All purification steps were done at 4 °C. The mycelium (20 g) was suspended in 60 ml buffer A (20 mM Tris/HCl pH 8·6, 1 mM EDTA, 20 mM KCl, 12% glycerol, 5 mM mercaptoethanol), and disrupted by sonication on ice (30 min total time, 30 s at amplitude 22 microns and 100 s pause; model Soniprep 150, MSE). Following centrifugation at 15000 g for 30 min, the supernatant was precipitated with ammonium sulfate to 50% (w/v) saturation, and the mixture was gently stirred for 1 h at 4 °C. The precipitate, obtained by centrifugation at 15000 g for 30 min, was dissolved in 12 ml buffer A, and dialysed overnight against the same solution. The sample was applied to a DEAE-cellulose column (25 ml; DE 52 Whatman) equilibrated with buffer A. After the column had been washed with the buffer, proteins were eluted with a linear gradient of KCl from 0 to 1 M in a total volume of 500 ml at a flow rate of 1 ml min-1. Fractions containing DNA-binding activity (24 ml) were pooled and dialysed overnight against buffer A. The dialysed sample was loaded onto a Heparin Sepharose CL-6B column (5 ml; Pharmacia). The column was washed with buffer A, and proteins were eluted with a linear gradient of KCl from 0 to 1 M in a total volume of 100 ml at a flow rate of 0·5 ml min-1. Fractions containing DNA-binding activity (8 ml) were pooled and dialysed overnight against buffer A. The dialysed sample was applied to a MonoQ HR 5/5 FPLC column (Pharmacia) equilibrated with buffer A. Proteins were eluted with a linear gradient (20 ml, 0–0·6 M KCl in buffer A). The active samples were dialysed overnight against buffer A and stored at -70 °C.

Protein analysis.
Protein concentrations were determined according to Bradford (1976 ), with BSA as a standard. Denaturing SDS-PAGE of proteins was done as described by Laemmli (1970 ), and gels were stained with Coomassie blue R250.

Preparation of radiolabelled DNA fragments.
A 253 bp fragment containing the sigF promoter region from -192 to +61 with respect to the tsp (Fig. 1a) was generated by PCR from plasmid pXSC2B (Potúcková et al., 1995 ) using a 5'-end-labelled oligonucleotide primer, mut53A, internal to sigF (5'-GAGGCGCAGTACTGGCCGGCACGG-3') and an unlabelled oligonuclotide, mut53B, from the upstream part (5'-GTTCATCACCGAACTGGTGAG-3'). The oligonucleotide was end-labelled with [{gamma}-32P]ATP [Amersham; 3000 Ci mmol-1 (111 TBq mmol-1)] and T4 polynucleotide kinase (Biolabs) as described in Ausubel et al. (1987 ). The labelled fragments were purified by PAGE as described in Ausubel et al. (1987 ).



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Fig. 1. Restriction map and nucleotide sequence of the S. aureofaciens sigF promoter region. (a) Restriction map of a 490 bp BclI–PvuII DNA fragment (open box) containing the sigF promoter region, and fragments used for binding studies. The black box represents the sigF gene, and the hatched box the upstream ORF1. The bent arrow denotes the position of the sigF-P2 promoter. The black bars below the map represent DNA fragments (5'-labelled at the end marked with an asterisk) that were used for binding studies. Numbers indicate nucleotide positions relative to the tsp of the sigF-P2 promoter. Relevant restriction sites are indicated. (b) Nucleotide sequence of the sigF promoter region. The deduced SigF protein product and ORF1 are given in a single-letter amino acid code in the second position of each codon, with the stop codon marked by an asterisk. The tsps of the sigF-P1 and sigF-P2 promoters are indicated by bent arrows. The proposed -10 and -35 boxes of the promoter are in bold characters and underlined. Positions of the oligonucleotide primers for PCR amplification of a 253 bp DNA fragment used for binding studies are designated by thin arrows above the sequence. The nucleotides that were protected from DNase I by RsfA binding are shaded. The numbers correspond to the nucleotide positions and refer to the published sequence of the S. aureofaciens sigF gene (Potúcková et al., 1995 ). The GenBank/EMBL/DDBJ accession number is M90413.

 
Gel mobility-shift assay.
The assays were performed essentially as described by Ausubel et al. (1987 ).32P-labelled DNA fragments (0·2 ng; 5000–10000 c.p.m.) were incubated with cell-free extracts or purified protein for 15 min at 30 °C in 15 µl total volume of the binding buffer, 2 µg sonicated salmon sperm DNA and 4·5 µg BSA. After incubation, protein-bound and free DNA was resolved on a non-denaturing polyacrylamide gel containing 4% acrylamide, 0·05% bisacrylamide and 2·5% glycerol, running (after 1 h prerun at 30 mA and 4 °C) in a high-ionic-strength buffer containing 50 mM Tris, 380 mM glycine and 2 mM EDTA, pH 8·5, at 4 °C at the same current. The gels were dried and exposed to an X-ray film.

DNase I footprinting.
Binding reactions were performed in 30 µl binding buffer, essentially under the same conditions as for the gel mobility-shift assays with 0·5 ng 32P-labelled DNA fragments (10000–30000 c.p.m.). After incubation for 15 min at 30 °C, 3 µl DNase I solution [5 U DNase I ml-1 (Boehringer Mannheim) in 100 mM MgCl2, 100 mM DTT] was added to the binding reaction. The reaction was incubated for 40 s at 37 °C, and stopped by 7·5 µl DNase I stop buffer (3 M ammonium acetate, 0·25 M EDTA, 0·1 mg tRNA ml-1), and extracted with 30 µl alkaline phenol/chloroform. An aqueous phase was precipitated with 3 vols ethanol. The resulting pellet, after washing with 70% ethanol and Speed Vac drying, was suspended in 5 µl Maxam loading buffer [80% formamide, 1 mM EDTA, 10 mM NaOH, 0·05% (w/v) bromphenol blue, 0·05% (w/v) xylene cyanole FF]. The DNA fragments were analysed on 6% DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980 ). After electrophoresis, the gels were dried and exposed to an X-ray film.

Expression of whiH in E. coli.
The S. aureofaciens whiH gene (Kormanec et al., 1999 ) was mutated to introduce a single NdeI site in the start codon using a mutagenic primer, mut87 (5'-CGGCCGACAAAGGACATATGAGTACCCTTGCG-3'). The gene was then cloned as a 900 bp NdeI–BamHI fragment in E. coli expression plasmid pET11c (Novagen) cut with the same restriction enzymes, resulting in plasmid pIJ-whiH1 for wild-type protein, and as a 900 bp NdeI–HindIII fragment in plasmid pET28a (Novagen), resulting in plasmid pET-whiH1 for the N-terminal His-tagged fusion protein. The DNA sequence of the fusion regions was verified by sequence analysis. The host strain for the pET series of expression plasmids, E. coli BL21(DE3)pLysS, transformed with the plasmids was grown in LB medium (Ausubel et al., 1987 ) containing 30 µg chloramphenicol ml-1 and 100 µg ampicillin ml-1 (for pET11c recombinants) or 40 µg kanamycin ml-1 (for pET28a recombinants) at 24 or 37 °C until OD600 0·5. Expression was induced with 1 mM IPTG. After 3 h (at 37 °C) or 14 h (at 24 °C), the cells were harvested by centrifugation at 12000 g for 10 min, and washed with ice-cold STE buffer. The pelleted cells were suspended in the binding buffer and disrupted by sonication. The cell lysates were centrifuged for 30 min at 30000 g and the supernatants were stored in aliquots at -70 °C.

Purification of His-tagged WhiH protein.
E. coli BL21(DE3)pLysS harbouring pET-whiH1 was grown in LB medium at 24 °C until OD600 0·5. After 14 h induction with 1 mM IPTG, the cells were harvested by centrifugation at 12000 g for 10 min, and washed with ice-cold STE buffer. The lysis of cells and native purification of His-tagged WhiH protein on His-Tag Bind resin (Novagen) was carried out as directed by the manufacturer. The eluted protein was dialysed overnight at 4 °C against 100 vols binding buffer, cleared by centrifugation at 30000 g for 10 min, and the supernatant was stored in aliquots at -70 °C.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Detection of sigF promoter-binding proteins in the course of differentiation by gel mobility-shift assays
Previously, we identified a gene, sigF, encoding an alternative {sigma}F of RNA polymerase that proved to have a role in the late stages of morphological differentiation in S. aureofaciens and S. coelicolor (Potúcková et al., 1995 ; Rezuchová et al., 1997 ). The gene was expressed only during sporulation, and it seemed to be indirectly dependent upon early sporulation sigma factor {sigma}RpoZ (Kormanec et al., 1996 ). Transcriptional control is often mediated by transcriptional factors that bind operator sites. Similarly, regulation of sigF expression might include some regulatory proteins activating or repressing sigF promoter activity. This could ensure temporal and spatial location of sigF expression during differentiation. To search for the presence of such transcriptional regulators, a gel mobility-shift assay was performed with the 253 bp sigF promoter fragment (region from -192 to +61 with respect to the tsp of the sigF promoter fragment; Fig. 1) and cell-free extracts from various developmental stages of S. aureofaciens grown on solid medium. Two distinct complexes were found, depending upon the developmental stage. A specifically retarded complex of identical mobility was found with cell-free extracts from substrate mycelium (13 h), and from the onset of aerial mycelium formation (19 h). A new complex of different mobility was identified during sporulation (36 h), which disappeared later in the process of spore maturation (60 h). The specificity of the interaction was demonstrated by the competitive binding of the unlabelled fragment by these proteins (Fig. 2a). The first complex is stronger (or more DNA-binding protein is present in the cell-free extract), since almost total shift was found with only 10 µg total proteins in the gel mobility-shift assay (Fig. 2b). With both cell-free extracts (from 13 and 19 h), two other weak retarded complexes of lower mobility were visible. These might correspond to a multimeric form of the protein bound in a complex of higher mobility, or some other proteins interacting with the promoter region. We tentatively named the protein that specifically bound the sigF promoter region in early stages (13 and 19 h) as RsfA (regulator of sigF), and the protein that caused the mobility-shift during sporulation (36 h) as RsfB.



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Fig. 2. Gel mobility-shift assays of a 253 bp DNA fragment containing the sigF promoter region from -192 to +61 with respect to tsp (Fig. 1) with cell-free extracts from S. aureofaciens. (a) Retardation of the 32P-labelled DNA fragment (0·2 ng) by 15 µg cell-free extracts from surface cultures of S. aureofaciens in the course of differentiation. S. aureofaciens was grown for 13 h (lanes 1 and 5), 19 h (lanes 2 and 6), 36 h (lanes 3 and 7) and 60 h (lanes 4 and 8) on solid Bennet medium (Horinouchi et al., 1983 ). These times correspond to the different stages of morphological differentiation: 13 h, vegetative substrate mycelium; 19 h, the onset of aerial mycelium formation; 36 h, aerial mycelium approximately at the time of septation; 60 h, spore maturation. Specificity of binding is illustrated by addition of 40 ng of the unlabelled 253 bp sigF promoter DNA fragment (lanes 1–4). (b) Retardation of the labelled fragment by increasing amounts of cell-free extracts from surface cultures of S. aureofaciens in the course of differentiation. Cells were harvested at the indicated time. Lane 1 contains the labelled fragment only. (c) Retardation of the labelled fragment by 15 µg cell-free extracts from S. aureofaciens grown in liquid minimal medium MNP (Hopwood et al., 1985 ) containing 0·5% (w/v) glucose (lanes 2–4) or mannitol (lanes 5–7) to different growth phases: exponential phase, 14 h (lanes 1 and 5); end of the exponential phase, 20 h (lanes 3 and 6); and stationary phase, 36 h (lanes 4 and 7). Lane 1 contains the labelled fragment only. The arrow indicates the free DNA fragment.

 
Gel mobility-shift assays with cell-free extracts from S. aureofaciens cultured in liquid media
Since S. aureofaciens is unable to sporulate in liquid medium, where it grows as substrate mycelium, we performed gel mobility-shift assays with the sigF promoter fragment (Fig. 1) and cell-free extracts from S. aureofaciens grown in liquid minimal medium NMP with 0·5% (w/v) carbon source. Since some developmental mutants are affected by glucose catabolic repression (Chater, 1998 ), we used two different carbon sources, glucose and mannitol. Substrate mycelium was harvested at three different growth phases (14 h – early-exponential phase; 20 h – late-exponential phase; and 36 h – stationary phase). A specifically retarded complex of identical mobility was found with cell-free extracts regardless of carbon source used (Fig. 2c). It corresponded to the complex found with cell-free extracts from solid-grown cells after 13 and 19 h (see below). The results of this shift assay indicate that the proposed RsfA protein is present mainly in young substrate mycelium. Its amount decreases in the course of growth, being almost undetectable in stationary phase. However, we can not rule out the possibility that it is present in all stages but that its binding activity decreases during growth.

Location of binding sites by gel mobility-shift assays using sigF promoter deletion fragments, and by DNase I footprinting
To map binding sites for presumed RsfA and RsfB proteins within the sigF promoter fragment, the 253 bp DNA fragment was digested with restriction endonucleases, and purified fragments (Fig. 1a) were used in gel mobility-shift assays with cell-free extracts from various developmental stages of S. aureofaciens grown on solid medium (Fig. 3). Assays with ApaLI-digested fragment (-25 to +61) showed that neither RsfA nor RsfB binds the fragment. The assays with BstBI-digested fragment (-90 to +61) revealed a binding activity of the RsfA protein, but not of RsfB. These results suggested differences in the binding of RsfA and of RsfB. Both proteins bind upstream of at least -25 bp. However, binding of RsfB is completely abolished in the BstBI-digested fragment, and binding of RsfA is almost not affected in this trimmed fragment. Thus it seems that the binding site of RsfA is in the fragment from -25 to -90 bp, and RsfB binds further upstream.



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Fig. 3. Mapping of the binding sites for the presumed RsfA and RsfB proteins within the sigF promoter fragment. The 253 bp DNA fragment containing the sigF promoter region and a series of fragments trimmed from the 5' end (see Fig. 1) were used in gel mobility-shift assays with 10 µg cell-free extracts from surface cultures of S. aureofaciens grown for 13 h (lanes 1, 4 and 7) and 36 h (lanes 2, 5 and 8) on solid Bennet medium. Lanes 3, 6 and 9 represent DNA fragments without cell-free extracts. Lanes: 1–3, the 86 bp ApaLI-digested fragment (-25 to +61); 4–6, the 152 bp BstBI-digested fragment (-90 to +61); 7–9, the original 253 bp DNA fragment containing the sigF promoter region (-192 to +61).

 
To locate RsfA and RsfB binding sites in the sigF promoter region, DNase I footprinting assays were carried out using the same 253 bp sigF promoter fragment (Fig. 1) and cell-free extracts from various developmental stages. Using the bottom strand from -192 to +61 bp with respect to the tsp of the sigF-P2 promoter, we found that the identical region from -87 to -25 bp upstream of the tsp was clearly protected with both cell-free extracts from 13 and 19 h, respectively (Fig. 4a). This is in agreement with deletion gel mobility-shift experiments, and suggests that the binding is probably caused by the same protein, RsfA, in both extracts. However, we were unable to locate the binding site of the proposed RsfB protein (present in sporulation), owing to a high endogenous nuclease activity in the 36 h cell-free extract (data not shown). Interestingly, the sequence of the RsfA-protected region includes two tandem repeats (5'-ATGCCC, 5'-CGATAT), and an inverted repeat (5'-ACGGTGC-N4-GCACAGT) overlapping a potential -35 region of the sigF-P2 promoter (Kormanec et al., 1996 ). These sequence motifs might be candidates for the RsfA binding.



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Fig. 4. DNase I footprints of S. aureofaciens cell-free extracts binding to the 32P-labelled 253 bp sigF promoter DNA fragment (0·5 ng) (Fig. 1). The vertical bars indicate the position of the RsfA binding site. The numbering is relative to the tsp of the sigF-P2 promoter. Lane 1 is without the cell-free extract. Lanes A and T represent G+A and C+T Maxam–Gilbert sequencing ladders, respectively (Maxam & Gilbert, 1980 ). (a) DNase I footprints with increasing amounts of cell-free extracts from surface cultures of S. aureofaciens in the course of differentiation. Cells were harvested at the indicated time. Lanes 2 through 4 contain 70, 170 and 340 µg cell-free extract. (b) DNase I footprints with increasing amounts of cell-free extracts from S. aureofaciens grown for 14 h in liquid minimal medium MNP (Hopwood et al., 1985 ) containing 0·5% (w/v) glucose or mannitol. Lanes 2 through 4 contain 10, 22 and 45 µg cell-free extract. All binding experiments were performed two to three times with independent sets of cell-free extracts and gave similar results.

 
To prove that the RsfA protein from the early stages of differentiation (13 and 19 h) is identical to the protein present mainly in young substrate mycelium of S. aureofaciens cultivated in liquid minimal medium NMP, we carried out DNase I footprinting assays using the same 253 bp sigF promoter fragment (Fig. 1) and cell-free extracts from 14 h cultivation with glucose or mannitol. Regardless of carbon source used, the same region from -87 to -25 bp upstream of the tsp was protected, as in the cell-free extracts from early developmental stages after 13 and 19 h (Fig. 4b). This is also consistent with the shift experiments, where the retarded complex of similar mobility was found with these cell-free extracts. This suggests that the same protein, RsfA, is responsible for the binding in cell-free extracts from liquid- and solid-grown cells.

Purification of RsfA
We tried to purify the RsfA protein from S. aureofaciens cultured for 14 h in liquid NMP medium with 0·5% (w/v) glucose by monitoring its binding to the sigF promoter fragment. Starting with 20 g mycelia and using the purification strategy described in Methods (ammonium sulfate fractionation, and chromatography on DEAE-cellulose, Heparin Sepharose and MonoQ FPLC), we found that the protein is extremely unstable, and its stability dramatically decreased with decreasing protein concentration. After the last chromatography step, it was almost inactive (gel-shift signals were visible after long exposure). Thus a substantial stabilization of the protein during the whole purification process is required for successful purification to homogeneity for N-terminal sequencing purposes. These experiments are in progress. However, during all purification steps, the RsfA protein was eluted in a single peak, though with decreasing binding activity. This indicates that the DNA-binding protein responsible for the sigF promoter fragment binding (RsfA) is a single protein. Moreover, DNase I footprinting with the fraction after DEAE-cellulose revealed the same protected region as with the cell-free extracts (data not shown).

Overexpression of whiH in E. coli, and gel-shift assays of purified WhiH with the sigF promoter
Given the dependence of sigF expression upon whiH in S. coelicolor (Kelemen et al., 1996 ), it seems plausible that a similar dependence also exists in S. aureofaciens, and this potential transcription factor WhiH might be a candidate for regulation of sigF expression. Moreover, similarly to in S. coelicolor, whiH expression in S. aureofaciens is developmentally regulated, with maximal activity during sporulation (Kormanec et al., 1999 ). Therefore, WhiH might correspond to the DNA-binding protein RsfB, which caused mobility shift during sporulation. We tried to prove this hypothesis by an investigation of a potential binding of WhiH to the sigF promoter. Therefore, the S. aureofaciens WhiH was overproduced in E. coli using a T7 RNA polymerase expression system. Plasmids pIJ-whiH1 (containing wild-type whiH) and pET-whiH1 (with 6xHis-tag fusion) were checked by sequencing, and successfully introduced into host strain E. coli BL21(DE3)pLysS. Total protein extracts, before and after induction with IPTG, were examined by SDS-PAGE. As shown in Fig. 5, prominent bands were clearly visible after induction with IPTG at 37 °C in the regions corresponding to the theoretical molecular mass of both WhiH (31·8 kDa) and His-tagged WhiH (34 kDa). After 3 h induction with IPTG at 37 °C, almost all WhiH was found in the insoluble fraction, and 6xHis–WhiH was partially in soluble form. When the culture temperature was decreased to 24 °C, almost all 6xHis–WhiH was in soluble form, and also about half of the WhiH was in the soluble fraction. Therefore, we purified the 6xHis–WhiH protein under native conditions (Fig. 5), and used it for gel mobility-shift assay with the sigF promoter fragment (Fig. 1). However, using the same binding conditions as for S. aureofaciens cell-free extracts, we did not detect any specific shift of the sigF promoter fragment. To check that the 6xHis tag did not influence the binding, we used cell-free extracts from E. coli containing both native WhiH (with plasmid pIJ-whiH1) or 6xHis-tagged WhiH (with plasmid pET-whiH1) for gel mobility-shift assays. Similarly, no shift was found using these cell-free extracts.



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Fig. 5. Overproduction and purification of WhiH in E. coli. Samples were analysed by SDS-PAGE (12·5% acrylamide). Lanes 1–4 contain crude extracts from E. coli BL21(DE3)pLysS carrying the corresponding plasmid, grown at 37 °C, and induced for 3 h with IPTG. Lanes: 1, pET11c; 2, pIJ-whiH1; 3, pET28a; 4, pET-whiH1. Lanes 5–7 contain samples from His-tagged WhiH protein purification from E. coli BL21(DE3)pLysS carrying pET-whiH1, grown at 24 °C, and induced for 14 h with IPTG. Lanes: 5, crude extract from induced cells carrying pET28a; 6, crude extract from induced cells carrying pET-whiH1; 7, soluble fraction from induced cells carrying pET-whiH1; 8, purified His-tagged WhiH protein after Ni2+-affinity chromatography. The arrow indicates the location of the WhiH protein. Lane S, molecular mass markers.

 

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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Developmental processes, such as sporulation, in bacteria require correct temporal and spatial expression of the genes necessary for the morphology and physiology of differentiated cells. This is mainly ensured by activation and inhibition of the key regulatory proteins – {sigma} factors of RNA polymerase. This process involves several levels, from inhibition and activation of transcription by transcriptional repressors and activators, respectively, to inhibition of the action of {sigma} factors by their antagonist, anti-sigma factors. Such orchestration at all regulatory levels ensures the correct developmental process. The {sigma}F factor of RNA polymerase is one of the key sporulation {sigma} factors in Streptomyces (Potúcková et al., 1995 ; Rezuchová et al., 1997 ). Transcriptional analysis of its gene, sigF, in S. aureofaciens has revealed that it is expressed only during sporulation, at the time when septation of aerial hyphae occurs, after 36 h growth on solid Bennet medium (Kormanec et al., 1996 ). By S1-nuclease mapping, we have found two very close tsps corresponding to putative promoters sigF-P1 and sigF-P2. However, the results of primer extension analysis in related strain S. coelicolor have revealed only the first tsp, corresponding to sigF-P2, though S1-nuclease mapping has revealed both tsps also in this strain (Kelemen et al., 1996 ). Thus it seems that the second tsp corresponding to sigF-P1 might arise from S1-nuclease over-digestion. Therefore, in the present study we refer only to the sigF-P2 promoter. Previous studies have also shown that the sigF promoter is dependent upon the early sporulation sigma factor {sigma}RpoZ (Kormanec et al., 1996 ). However, this dependence is very likely indirect, since the sigF promoter did not contain {sigma}RpoZ consensus sequence (Kormanec et al., 1999 ). Thus the sigF promoter is likely recognized by a holoenzyme of RNA polymerase containing another {sigma} factor. This temporal, and probably also spatial, transcriptional activation of sigF expression seems to be a key regulatory event in the process of Streptomyces sporulation, similar to the activation of prespore-specific {sigma}F in B. subtilis (Kroos et al., 1999 ). It is likely that some transcriptional activators or repressors may participate in this process. To investigate this possibility was the main task of the present paper. The results demonstrate a possible participation of such proteins in the regulation of sigF expression in S. aureofaciens. Investigation of DNA-binding proteins in the course of differentiation actually revealed the presence of two putative proteins, RsfA and RsfB. While RsfA bound the sigF promoter during early stages of differentiation (13 and 19 h), RsfB bound the promoter later during sporulation, approximately at the time of aerial mycelium septation into spores (at 36 h). Considering the results of gel mobility-shift assays with deleted sigF promoter fragments and DNase I footprinting, it seems that the DNA binding is caused by two independent proteins, RsfA and RsfB. However, this question would be fully answered after isolation of both proteins, investigation of their binding to the sigF promoter, and their N-terminal sequencing. Our preliminary experiments with the isolation of the potential repressor-like protein RsfA to homogeneity failed owing to its high instability in the more purified form. The purification of RsfA for N-terminal sequencing purposes, and cloning of the gene encoding this protein, are in progress.

Given the course of sigF expression, it is likely that the binding of RsfA might switch off the promoter in the early stages of differentiation, where its expression is not required. This potential repressor-like function of RsfA is also indicated by DNase I footprinting analysis, where the protein was found to protect a region that overlaps the proposed -35 region of the sigF promoter. At the time of sporulation, probably when sporulation septa are formed, the activation of sigF might be ensured by an activator, probably RsfB. For this type of sigF transcriptional regulation, it is possible that the sigF promoter might be recognized by a holoenzyme of RNA polymerase containing a {sigma} factor that is active during all stages of differentiation, like the principal sigma factor {sigma}HrdB, for instance (Kormanec & Farkasovský, 1993 ). Interestingly, the putative -10 region of the sigF-P2 promoter (TGTGAT) shows limited similarity to the -10 consensus sequence (TAgPuPuT) for {sigma}HrdB-dependent promoters (Strohl, 1992 ). However, the sequence around the -35 region (with the spacer of 16–18 nt) shows no obvious similarity to the corresponding consensus sequence (TTGACN) of the same class of promoters (Fig. 1b). In E. coli, promoters with poor similarity to the consensus in the -35 region are frequently positively controlled by activators that bind upstream of the -35 region, and make specific contacts with the holoenzyme of RNA polymerase, thereby activating the process of transcription in various steps (Gralla, 1990 ). Thus the holoenzyme of RNA polymerase containing {sigma}HrdB might be a candidate for directing the sigF promoter. Of course, to prove this assumption needs further experimentation.

Based on the developmental regulation of whiH expression in S. aureofaciens (Kormanec et al., 1999 ), and assuming that S. aureofaciens sigF expression is dependent upon sporulation-specific transcription factor WhiH, as found in S. coelicolor (Kelemen et al., 1996 ), it raises the possibility that the putative RsfB could be WhiH. However, the results of the binding experiments with the overproduced WhiH in E. coli (with or without N-terminal fusion with 6xHis tag) indicate that this dependence is probably indirect. However, we can not rule out that the failure of E. coli-overproduced WhiH binding was caused by an absence of some WhiH modification or an effector molecule, present in S. aureofaciens, but absent in E. coli.

Summarizing all these results, we suggest a proposed model of regulation of sigF in S. aureofaciens. During differentiation on solid medium, the putative repressor RsfA binds to the sigF promoter and hinders the access of RNA polymerase to the promoter. This binding is relieved by an unknown mechanism after the septation of aerial hyphae into spore compartments. At that time, the putative activator RsfB is synthesized or activated, and binds to the sigF promoter, which activates transcription by RNA polymerase holoenzyme containing the {sigma} factor that is present in all stages of differentiation (probably the principal {sigma}HrdB factor). This ensures the temporal and compartment-specific expression of sigF in the spore compartment, where after translation {sigma}F could direct the expression of sigF-regulon-encoding proteins required for spore maturation. Of course, this model is preliminary, and the activation of {sigma}F might also involve an anti-sigma factor, or other mechanisms, similar to the activation of {sigma}F in B. subtilis (Kroos et al., 1999 ).

Using the gel mobility-shift assay, we also tried to investigate sigF expression in the genetically best studied S. coelicolor A3(2) strain. However, using cell-free extracts from liquid-grown S. coelicolor in the same minimal medium, NMP, no retardation was detected with any extract from different growth phases. Using cell-free extracts from solid-grown cells on minimal MM medium with 0·5% (w/v) mannitol during differentiation, only a weak retarded fragment was identified after 1 d growth (substrate mycelium) (J. Kormanec, unpublished results). Interestingly, the comparison of sigF promoters from S. coelicolor and S. aureofaciens has revealed that, despite the high sequence similarity between these two promoters up to position -58, the S. coelicolor sigF promoter lacks a large part of the proposed RsfA-protected region (Fig. 6). Thus though regulation of differentiation has several common features between these strains (Kormanec et al., 1994 , 1996 , 1998 , 1999 ), there are some regulatory circuits that are specific for each strain.



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Fig. 6. Comparison of the S. aureofaciens (SigFSa) and S. coelicolor (SigFSc) sigF promoter sequences. Whole intergenic regions from the stop codon of the upstream ORF1 to the initiation codon of sigF (both in bold and underlined) were compared. The tsps of the sigF promoters are indicated by bent arrows. The proposed -10 and -35 boxes of the promoters are in bold and underlined. Inverted repeats are designated by thin arrows. Nucleotides in the S. aureofaciens sigF promoter region which were protected from DNase I by RsfA binding have a black background. Numbers correspond to the nucleotide positions and refer to the published sequences of the S. aureofaciens and S. coelicolor sigF genes (Potúcková et al., 1995 ).

 

   ACKNOWLEDGEMENTS
 
We would like to thank Mrs Renáta Knirschová for excellent technical assistance. We are grateful to Dr Eva Kutejova for her help with purification of RsfA. This work was supported by Grant 2/7001/20 from the Slovak Academy of Sciences.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 31 May 2000; revised 26 July 2000; accepted 31 July 2000.



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