Folia Microbiol. 46 (6), 527 534 (2001)
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Identification of DNA-Binding Proteins Involved in Regulation of Expression of the Streptomyces aureofaciens whiH Gene Encoding a Sporulation Transcription Factor D. HOMEROVA, J. KORMANEC* Institute of Molecular Biology, Slovak Academy of Sciences, 842 51 Bratislava, Slovakia fax +421 2 5477 2316 e-mail
[email protected] Received 26 July 2001 Revised version 22 August 2001 ABSTRACT. Using the gel mobility-shil%assay with protein fractions from different developmental stages of solid-grown Streptomyces aureofaciens, we identified two different proteins specifically bound to the whiH promoter region. Only one protein (RwhA) was detected in young substrate mycelium cultivated in liquid medium. On comparing the mobility of the resulting complexes, one of the bound proteins present in substrate mycelium and in early stages of aerial mycelium seemed to be identical with the RwhA. The other detected protein with a higher mobility (RwhB) was present in all developmental stages except for mature spores. DNA footprinting analysis localized the binding site of RwhB to nucleotides -23 to +40 relative to the transcription start point of the PwhiHpromoter. RwhA from young substrate mycelium protected the DNA frag-
ment from -106 to -77 in coding strand and -126 to -82 in noncoding strand. WhiH has homology to a large family of metabolism-related repressors and seems to regulate negatively its own expression. These observations (and the results of transcription analysis of the whiH gene obtained earlier) suggest that two different proteins influence the expression of whiH gene in S. aureofaciens. The putative repressor-like RwhA protein protects expression of whiH in substrate mycelium either in liquid medium or during differentiation. The other detected protein, RwhB, which binds to the whiH promoter region during differentiation, may represent two forms of WhiH, one with a repressor role at the beginning of differentiation and second with the role of activator at the time of sporulation.
Streptomycetes are Gram-positive soil bacteria undergoing an exceptional process o f morphological differentiation, phenotypically similar to the sporulation o f eukaryotic filamentous fungi. It involves the formation o f spore-bearing aerial hyphae on mycelial colonies. The process o f differentiation is regulated at several levels, o f which heterogeneity o f o--factors o f RNA polymerase plays an important role (Chater 1998). At least three R N A polymerase o- factors are proposed to be involved in the regulation o f differentiation; o-whiG in Streptomyces coelicolor (Chater et al. 1989) and o-rpoZ in S. aureofaciens (Kormanec et al. 1994) are crucial in initiation o f sporulation, o-P is essential in the late stage o f spore formation in both species (Potfi~kovzi et al. 1995; l~e2uchov~i et al. 1997), and the recently identified o-bldN is essential for aerial mycelium formation (Bibb et al. 2000). Several regulatory genes important in the regulation o f this process have been identified but detailed mechanism o f the regulation o f differentiation in streptomycetes remains unknown. Two mutant classes, bld (defective in aerial hyphae formation) (Merick 1976) and whi (defective in spore formation) (Chater 1972), have been studied in the genetically best-studied S. coelicolor A3(2) strain. w h i H is one o f several known early sporulation whi genes which are necessary for normal spore formation (Chater 1972; McVittie 1974). The deduced protein WhiH is homologous to a large GntR family o f transcriptional factors. Transcription o f w h i H is initiated at a single promoter and is developmentally regulated. Transcript levels are increased in the w h i H mutant, indicating some direct or indirect autoregulatory mechanism. Expression o f the w h i H gene depends directly on sporulation-specific o- factor, o"WhiG, which is involved in the regulation o f sporulation (Ryding et al. 1998). Differentiation o f S. coelicolor has several common features with that o f S. aureofaciens despite some morphological differences between these two species. Though several regulatory proteins are common, the regulation o f differentiation has some specific features in each species (Kormanec et al. 1994, 1996, 1998, 1999). in S. aureofaciens, the w h i H gene encoding a regulatory protein was identified. The deduced WhiH protein had 83 % identity with WhiH from S. coelicolor and homology to the GntR family o f transcription regulators, as revealed in S. coelicolor. Similarly to w h i H from S. coelicolor, the w h i t t promoter is induced at the time o f aerial mycelium formation and is active till the end o f sporulation with the maximum *Corresponding author.
528 D. HOMEROVA and J. KORMANEC
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of activity at the time of septation of aerial hyphae. No WhiH expression has been shown in S. aureofaciens rpoZ mutant; this indicates a dependence of S. aureofaciens PwhiHupon sporulation specific cr factor ~rpoZ. Although the putative -35 and -10 regions of whiH promoters in both species are identical, there are some differences in the positions of inverted repeats, which indicates that there might be some differences in the regulation of the two promoters (Kormanec et al. 1999). Here we investigated proteins which specifically bind to the S. aureofaciens whiH promoter region in the course of differentiation. Their binding sites were determined by DNAaseI footprinting and found to overlap with the whiH promoter region. We used the WhiH protein overproduced in E. coli to explore its binding to the whiH promoter region.
M A T E R I A L S AND M E T H O D S
Bacterial strains, plasmids and culture conditions. S. aureofaciens wild type strain CCM3239 was from Czechoslovak Collection of Microorganisms (Brno, Czechia). For cell-free extracts preparation, in amount of 108 spores was inoculated into 20 mL of!iquid medium NMP (Hopwood et al. 1985) containing different carbon sources at 0.5 % (W/V) final concentration, and cultured at 30 ~ to the early exponential phase (14 h), late exponential phase (20 h), and stationary phase (36 h). For surface cultures, 108 spores were spread on sterile cellophane membranes placed on Bennet medium (Horinouchi et al. 1983) and grown for 13 h (substrate mycelium), 19 h (the beginning of aerial mycelium formation), 36 h (aerial mycelium approximately at the time of septation), and 60 h (spore maturation). DNA manipulations in E. coli were done according to Ausubel et al. (1995), and those in Streptomyces according to Hopwood et al. (1985). DNA fragments were isolated from agarose gel (Kormanec et al. 2001); nucleotide sequencing was performed by the chemical method (Maxam and Gilbert 1980). Preparation of cell-free extracts. Liquid-grown mycelium was harvested by centrifugation (12 000 g, 10 rain) and washed by ice-cold STE buffer (in retool/L: Tris-HCI 10, NaCI 100, EDTA 1; pH 8). Solidgrown mycelium was scraped off the cellophane. All successive steps were carried out at 4 ~ 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 suspended in binding buffer (in retool/L: Tris-HC1 12.5, KC1 60, EDTA 1, 1,4-dithiothreitol 1; 12 % (W/V) glycerol; pH 7.9), and cell debris was removed by centrifugation (30 000 g, 30 rain). The cell-free extracts were stored in aliquots at -70 ~ and used in gel mobility shift assays and DNase I footprinting experiments. Protein concentration was determined according to Bradford (1976), with bovine serum albumin as standard. Preparation of labeled DNA fragments. A 393-bp Sau3AI-NotI DNA fragment bearing the promoter region ofS. aureofaciens whiH gene, and 336-bp AgeI-BsiWI DNA fragment bearing promoter region of the S. coelicolor whiH gene (Fig. 1) were 5'-end-labeled with y-32p-ATP (Amersham; 111 TBq/mmol) and T4-polynucleotide kinase (Biolabs) (Ausubel et al. 1995). A 240-bp fragment containing the whiH promoter region from -202 to +38 with respect to the transcription start point (tsp) (Fig. 1A) was generated by PCR using primer mutwhiH1 internal to whiH (5'-GGG CGG CGG TCA TCA TGG TGT G-3') and oligonuclotide mutwhiH2 from the upstream part (5'-GAT CGT TCC GGT GCG GGA GGC G-3') (Fig. 2). For preparing radiolabeled probe, one of the oligonucleotides (either mutwhiHlor mutwhiH2) was first 5'-end-labeled with y-32p-ATP (Amersham; 111 TBq/mmol) and T4-polynucleotide kinase (Biolabs) (Ausubel et al. 1995). The radiolabeled fragments were purified by PAGE (Kormanec et al. 2001). Gel mobility-shift assays were performed essentially according to Ausubel et al. (1995). 32p-Labeled DNA fragments (0.2 rig, 300-600 kBq) were incubated with cell-free extracts or purified protein (15 min, 30 ~ in the binding buffer (15 laL total volume), 2 lag sonicated salmon sperm DNA, and 4.5 lag BSA. After incubation, protein-bound and free DNA were resolved on nondenaturing polyacrylamide gel containing (in %, W/V) acrylamide 4, bis-acrylamide 0.05 and glycerol 2.5, running (after a 1-h pre-run at 30 mA and 4 ~ in a high-ionic-strength buffer (in retool/L: Tris 50, glycine 380, EDTA 2; pH 8.5) at 4 ~ at the same current. The gels were dried and exposed to an X-ray film. DNAaselfootprinting. Binding reactions were performed in 30 laL of binding buffer, essentially under the same conditions as for the gel mobility shift assays with 5 ng of the 32p-labeled DNA fragments (0.6-1.8 MBq). After incubation for 15 min at 30 ~ 3 ILL of DNAase I (Boehringer Mannheim) solution (5 U/mL of DNAase I in 100 mmol/L MgCI2, 100 mmol/L 1,4-dithiothreitol) was added to the binding reaction. Reaction mixture was incubated (40 s, 37 ~ and the reaction was stopped by 7.5 laL of DNAase 1 stop buffer (3 mol/L ammonium acetate, 0.25 mol/L EDTA, 0. I g/L tRNA). The mixture was extracted with 30 ~tL of alkaline phenol-chloroform mixture. An aqueous phase was precipitated by 3 volumes of ethanol.
REGULATION OF EXPRESSION OF THE S. aureofaciens whiH GENE
2001
529
After washing with 70 % (V/V) ethanol and Speed Vac drying, the resulting pellet was suspended in 5 gL Maxam loading buffer (80 %, V/V, formamide; 1 mmol/L EDTA; l0 mmol/L NaOH; 0.05 % (W/V) bromophenol blue; 0.05 % (W/V) xylene cyanole FF). The DNA fragments were analyzed on 6 % DNA sequencing gels together with G + A and T + C sequencing ladders derived from the end-labeled fragments (Maxam and Gilbert 1980). After electrophoresis, the gels were dried and exposed to an X-ray film. A 100 bp
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Fig. i. Chromosomal DNA comprising whiH promoter regions (open boxes) and fragments used for binding studies; the dark boxes represent the whiH gene; the bent arrows represent direction of the apparent transcript from PwhIH; the black bars below the maps represent the DNA fragments (5'-labeled at the end marked with an asterisks) that were used for binding studies; relevant restriction sites are indicated; numbers indicate nucleotide positions relative to tsp of the PwhiH promoter, A: Restriction map of a 500-bp DNA fragment of S. aureofaciens. B: Restriction map of a 400-bp DNA fragment o f S coelicolor; the hatched box represents the upstream ORFI.
RESULTS AND DISCUSSION
Detection of whiH promoter-binding proteins during differentiation by gel mobility-shift assay The transcription factor WhiH is one of the proteins necessary for normal sporulation of Streptomyces spp. and its role in sporulation is complex. Inactivation of whiH does not fully block all aspects of spore chain development (Fl~irdh et al. 1999). A low level of transcription of the late sporulation genes sigF and whiE was detected in whiH point mutant (Kelemen et al. 1996, 1998). This can indicate that at least one other regulatory element must be closely involved in activating the late sporulation genes. WhiH as a DNAbinding protein can have a direct positive role in morphological development, or it can act as repressor of another negative regulator which prevents differentiation. To understand the regulation of whiH expression in more detail, we tried to identify the proteins involved in the regulation of whiH transcription. Previously we identified the whiH gene encoding the putative sporulation transcription factor WhiH essential for differentiation in S. aureofaciens (Kormanec et al. 1999). To study the regulation of expression of whiH and to explore the possible role of WhiH in its own regulation in S. aureofaciens, gel-mobility shift assay was performed with a promoter fragment (Figs 1 and 2) and cell-free extracts from various developmental stages ofS. aureofaciens grown on a solid medium. More distinct complexes were detected, depending upon developmental stage (Fig. 3A). Two specifically retarded complexes of similar mobility were found with cell-free extracts from substrate mycelium (13 h), and from the onset of aerial mycelium formation (19 h). During sporulation (36 h), only one complex (with higher mobility) was identified, which was substantially reduced later during spore maturation (60 h). We consi-
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D. HOMEROV,~ and J. KORMANEC
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dered this protein with lower mobility that specifically bound to the PwhiH promoter region to be a regulator of whiH (RwhA), and the protein with higher mobility as RwhB. To compare the binding of these proteins to the P~hi/~' promoter regions from S. aureofaciens and S. coelicolor, a similar gel mobility-shift assay was performed using whiH promoter region from S. coelicolor (Fig. 1B). As shown in Fig. 3B, only one shift (corresponding to RwhB) was detected at the beginning of aerial mycelium (19 h) and during sporulation (36 h) (lanes 3 and 4). According to the gel mobility-shift assay with cell-free extracts from solid and liquid media (see below) at least two proteins (RwhA and RwhB) bind to the whiH promoter region. The RwhA that was found in substrate mycelium and in the beginning of aerial mycelium seems to be identical to that detected in the exponential phase of liquid-grown substrate mycelium (14 h). It could represent a protein which negatively influences the expression of the whiH gene, since whiH is not expressed in liquid media during substrate mycelium and early stages of aerial mycelium. The second protein, RwhB, was detected both at the time when whiH is not expressed (substrate mycelium and beginning of aerial mycelium) and at the time when whiH is expressed (sporulation). To explain this result we needed to consider the "two roles" of the WhiH protein as suggested by Flgrdh et al. (1999). WhiH resembles a family of DNA-binding regulatory proteins
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REGULATION OF EXPRESSION OF THE S. aureofaciens whiH GENE 531
responsive to carboxylate-containing intermediates of carbon metabolism. It can sense some metabolic intermediates that change their concentration during the development o f streptomycetes, Aerial hyphae are formed on substrate mycelium that undergoes lysis, and the metabolic components produced support the growth
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Fig. 3. Gel mobility-shift assays of the DNA fragment containing the whiH promoter region with cell-free extracts from S. aureofaciens. A: Retardation of the 240-bp S. aureofaciens 32p-labeledDNA fragment (0.2 ng) from -202 to +38 with respect to tsp by 15 lag of cell-free extracts from surface cultures of S. aureofaciens during differentiation; lane 1 contains the labeled fragment only; S. aureofaciens grew 13 b (2), 19 b (3), 36 h (4), and 60 h ('5-)on solid Bennet medium (Horinouchi et al. 1983) which corresponds to the different stages of morphological differentiation (13 h - vegetative substratc mycelium, 19 h - the onset of aerial myeelium formation, 36 h - aerial mycelium approximately in the time of septation, 60 b - spore maturation). B: Retardation of the 336-bp S. coelicolor labeled DNA fragment (0.2 rig) by 15 lag of cell-freeextracts from surface cultures during differentiation;lane 1 contains the labeled fragment only, in lanes 2-5 are cell-free extracts from the same cultivation periods as in lanes 2-5 of Fig. 2A. C:. Retardation of the 240-bp S. aureofaciens 32p-labeled DNA fragment (0.2 ng) from -202 to +38 relative to tsp by 15 lag of 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 (5-7) to different growth phases: 14 h - exponential phase (2, 5), 20 b - end of the exponential phase (3, 6), 36 h - stationary phase (4, 7); lane 1 contains the labeled fragment only; arrows indicate free DNA fragment.
o f the aerial hyphae (Mendez et al. 1985). A change o f the concentration o f metabolic intermediates could give rise to two forms o f WhiH. One, acting as a repressor o f some sporulation genes, should prevent an earlier onset o f sporulation and could repress also its own expression. The other whose proportion increases at later stages and is called WhiH*, could directly or indirectly positively regulate sporulation processes by increased expression o f genes necessary for sporulation septation and maturation o f spores. We assume that WhiH represses its own expression at the earlier stage of differentiation (substrate mycelium and beginning o f aerial mycelium) and, later, the second form, WhiH*, activates its own expression (developed aerial mycelium). However, the gel mobility-shift assay with WhiH expressed in E. coli or with purified WhiH (Homerovfi et al. 2000) did not show any binding to the w h i H promoter region; this can be explained by lack o f some necessary modification o f WhiH, which is present in S. aureofaciens but not in E. coli (unpublished results). Gel mobility-shift assay using w h i H promoter region from the genetically best-studied S. coelicolor A3(2) revealed a similar binding o f RwhB protein in cell-free extracts from solid-grown cells. This may be explained by assuming that the bound protein is WhiH; this protein from both strains (S. c o e l i c o l o r and S. aureofaciens) exhibit a high similarity and the DNA-binding regions are identical (Kormanec et al. 1999). However, the binding o f a second protein with lower mobility (RwhA) was not confirmed in S. coelicolor. Gel mobility-sh(ft assay with cell-free extracts f r o m S. aureofaciens c u l t u r e d in liquid m e d i u m Since S. aureofaciens is unable to s p o r u l a t e i n liquid medium (where it grows as substrate mycelium), we performed a gel mobility-shift assay with the promoter fragment (Fig. 1A) and cell-free extracts the strain grown in a liquid minimal medium. Since some developmental mutants are affected by glucose cata-
532 D. HOMEROVA and J. KORMANEC
Vol. 46
bolite repression (Chater 1998), we used two different carbon sources, glucose and mannitol. A specifically retarded complex of identical mobility was found with cell-free extracts after 14 h of growth regardless of the carbon source used (Fig. 3C). It corresponded to the complex with RwhA found with cell-free extracts from solid-grown cells after 13 and 19 h. As shown in lanes 3 and 6 of Fig. 3C (20 h, late exponential phase), this complex was substantially reduced and a new weak complex with a higher mobility was visible with cell-free extracts from the stationary phase of growth (36 h) especially when mannitol was used as carbon source. The results of this shift assay indicate that the putative regulatory protein RwhA is present mainly in young substrate mycelium and its amount decreases during growth, or that it is present in all stages but its binding activity decreases during growth. The weaker complex with the higher mobility present only in stationaryphase cells might correspond to RwhB. Considering the results of gel mobility-shift analysis and DNA footprinting (see below), it seems that both identified proteins (RwhA and RwhB) coordinately regulate whiH expression. In liquid media and in the early stages of differentiation, when expression of the whiH gene is not required, repressor-like RwhA protein binds to the upper part of whiH promoter region and prevents the expression of whiH gene. In substrate mycelium and at the very beginning of aerial mycelium formation, another protein, RwhB, that might correspond to one form of WhiH, ensures further inhibition of whiH expression by binding to the site overlapping -35 and -10 region of the whiH promoter. As the concentration of some intermediate of carbon metabolism increases, WhiH may be modified and its active form WhiH* switches on the promoter. This model is preliminary and further investigations are necessary to be performed. Location o f binding sites by DNAase I footprinting To locate binding the sites in the whiH promoter region, DNAase I footprinting assay was carried out with the 393-bp whiH promoter fragment (Fig. 1A) and cell-free extracts from various developmental stages. Using the bottom strand from -202 to +191 bp with respect to the tsp of whiH promoter, we found that the identical region from -23 to +40 bp was protected with both cell-free extracts from the 13th and 19th h, respectively (Fig. 2 and 4A). However, we were unable to locate the binding site of the proposed protein present during sporulation, owing to a high endogenous nuclease activity in a 36-h cell-free extract (data not shown). To demonstrate that the protein from the early stages of differentiation (13 and 19 h) is different from the protein present mainly in substrate mycelium of S. aureofaciens cultivated in liquid minimal medium NMP, we carried out DNAase I footprinting assay using the 240-bp PCR whiH promoter fragment (Fig. 1A) and cell-free extracts from 14 h cultivation with glucose. According to the gel mobility-shift assays, this cell-free extract contains only RwhA (Fig. 3C). We used the shorter fragment to determine the binding region more upstream from tsp, which we were unable to detect with longer (393 bp) fragment. Using both strands we recognized the binding region, which was different from that recognized with cell-free extracts from various developmental stages (13 and 19 h). The binding region was from -106 to -77 for the coding strand and from -126 to -82 for the noncoding strand (Fig. 2 and 4B). Since this cell-free extract contains only RwhA (Fig. 3C), it is plausible to assume that this binding site corresponds to RwhA, and the binding site identified with cell-free extracts from early stages of differentiation corresponds to RwhB. Previously, we identified DNA-binding proteins involved in the regulation of expression of the S. aureofaciens sigF gene, encoding ~ factor ~F with a role in spore maturation (Homerov~i et al. 2000). One of the proteins (designated RsfA) with putative repressor function was present in early stages of differentiation (13 h and 19 h) and in a young mycelium from a liquid culture. It strongly resembles RwhA binding to the whiH promoter, identified by gel mobility-shift assay in solid-grown and liquid-grown mycelium. Although the comparison of protected nucleotide sequences in both whiH and sigF promoters revealed some similarity (Fig. 5), it is not yet proved whether RwhA corresponds to RsfA. If it does, then RsfA might constitute a general repressor for sporulation genes. It will be necessary to purify the two proteins (RsfA and RwhA) to homogeneity and to perform the binding studies with both promoters using the purified protein. This work was supportedby grant no. 2/7001/21 fromthe SlovakAcademyof Sciences.
REGULATION OF EXPRESSION OF THE S. aureofaciens whiH GENE
2001
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Fig. 4. DNAase 1 footprints of S aureofaciens cell-tree extracts binding to the 32p-labeled S. aureofaciens whiH promoter DNA fragment (5 rig); vertical bars indicate the position of RwhA- and RwhB-binding sites; the numbering is relative to the tsp of the PwhiH promoter; lane f is without the cell-free extract; lanes A and T represent G + A and C + T Maxam-Gilbert sequencing ladders, respectively (Maxam and Gilbert 1980). Left: DNAase I footprints with an increasing amount of cell-free extracts from surface cultures orS. aureofaciens during differentiation using the 393-bp PwhiHpromoter fragment (see Fig. IA); cells were harvested at the indicated time points; lanes 2 and 3 contained 50 and 200 p.g of cell-free extracts, respectively. Right: DNAase I footprints with increasing amount of cell-free extracts from S aureofaciens grown 14 h in liquid minimal medium MNP (Hopwood et al 1985) containing 0.5 % glucose using 240 bp PwhtHpromoter fragment (see Fig. IA); lanes 2 and 3 contained 50 and 200 ~g of cell-free extracts, respectively; CS - coding strand, NS - noncoding strand. All binding experiments were performed 2-3 times with independent sets of cell-free extracts and similar results were obtained.
whiHp sigFp Fig. 5. Comparison of the S. aureofaciens whiH and sigF promoter sequences protected from DNAase 1 by binding of proteins; in the whiH promoter, the RwhA-protected sequence from upstream region was compared with the stgF promoter sequence protected by RsfA binding (Homerova et aL 2000); identical nucleotides are boxed.
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D. HOMEROV,~ and J. KORMANEC
Vol. 46
REFERENCES
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