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
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ABSTRACT |
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Keywords: Streptomyces aureofaciens, differentiation, repressor, promoter, sigma factor
Abbreviations: tsp, transcription start point
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INTRODUCTION |
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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 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
factors (Kroos et al., 1999
). Two genes encoding
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 (Potú
ková et al., 1995
;
e
uchová 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
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
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
factor encoded by whiG/rpoZ is indirect, and the sigF promoter is probably recognized by a holoenzyme of RNA polymerase containing some other
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.
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METHODS |
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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, 00·6 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 (Potú
ková 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 [
-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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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 (1000030000 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 NdeIBamHI 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 NdeIHindIII 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.
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RESULTS |
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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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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 6xHisWhiH was partially in soluble form. When the culture temperature was decreased to 24 °C, almost all 6xHisWhiH was in soluble form, and also about half of the WhiH was in the soluble fraction. Therefore, we purified the 6xHisWhiH 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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DISCUSSION |
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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 factor that is active during all stages of differentiation, like the principal sigma factor
HrdB, for instance (Kormanec & Farka
ovský, 1993
). Interestingly, the putative -10 region of the sigF-P2 promoter (TGTGAT) shows limited similarity to the -10 consensus sequence (TAgPuPuT) for
HrdB-dependent promoters (Strohl, 1992
). However, the sequence around the -35 region (with the spacer of 1618 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
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 factor that is present in all stages of differentiation (probably the principal
HrdB factor). This ensures the temporal and compartment-specific expression of sigF in the spore compartment, where after translation
F could direct the expression of sigF-regulon-encoding proteins required for spore maturation. Of course, this model is preliminary, and the activation of
F might also involve an anti-sigma factor, or other mechanisms, similar to the activation of
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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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 May 2000;
revised 26 July 2000;
accepted 31 July 2000.
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