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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. σH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of σH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and σH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of σH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In unicellular prokaryotes, chromosome duplication is typically followed by formation of the division septum, cytokinesis and cell separation. However, in vegetatively growing Streptomyces, chromosome duplication is not followed by septation and cell separation. This generates a substrate mycelium of branching hyphal filaments that grows across and into the agar. Later, presumably in response to nutrient limitation, the substrate mycelium supports the development of specialized aerial hyphae that grow out of the aqueous environment of the substrate mycelium and into the air (Kelemen and Buttner, 1998), a phase of differentiation that requires the action of a number of ‘bald’ (bld) developmental regulatory genes, one of which, bldD (Merrick, 1976; Elliot et al., 1998), is relevant to the work described here. When extension of the aerial hyphae stops, their multigenomic tips undergo synchronous, multiple septation to give rise to unigenomic prespore compartments (Schwedock et al., 1997; Chater, 1998).

Localized morphological differences within the ‘tissues’ of the mycelial Streptomyces colony suggest that each has its own developmental programme. The spatial pattern of expression of individual genes in the different ‘tissues’ can now be defined by transcriptional fusions to an enhanced variant of the green fluorescent protein (EGFP). By this means, transcription of sigF (encoding the late spore-specific sigma factor σF; Potúčkováet al., 1995) and transcription from one of two ftsZ promoters were shown to be restricted to sporulating aerial hyphae (Sun et al., 1999; Flärdh et al., 2000). Conversely, transcription of redD, encoding a pathway-specific activator for the biosynthetic genes specifying the antibiotic undecylprodigiosin, was restricted to the substrate hyphae (Sun et al., 1999), the site of undecylprodigiosin biosynthesis. The hyphae forming these distinctive ‘tissues’ may be subject to different environmental and metabolic stress conditions.

In the unicellular bacterium, Bacillus subtilis, diverse stresses, such as heat shock, salt stress and ethanol, induce a common set of proteins, called the general stress proteins (GSPs; Hecker et al., 1996; Hecker and Völker, 1998; Völker et al., 1999; Price, 2000). In this organism, the general stress response is controlled by σB (Price, 2000), and the vast majority of GSP genes (> 100; Hecker et al., 1996; Völker et al., 1999) are induced from σB-dependent promoters. In addition to these non-specific GSPs, each stress induces a set of specific proteins that may confer specific protection. In contrast, two-dimensional gel analysis of changes in the Streptomyces coelicolor proteome after induction with various stresses shows that such treatments induce mutually exclusive stimulons (Vohradsky et al., 1997; 2000). Importantly, many osmotic shock and heat shock proteins are transiently induced during growth in liquid medium and differentiation on solid medium in Streptomyces (Bucca et al., 1995; Puglia et al., 1995; Vohradsky et al., 1997; 2000).

Recently, we identified three paralogous sigma factors (σH, σI and σJ), closely related to Bacillus subtilisσB, that are associated with stress responses in S. coelicolor. σH was identified biochemically through its ability to direct in vitro transcription of a heterologous promoter, ctc, which is part of the σB general stress response regulon in B. subtilis (Viollier et al., submitted). When introduced into S. coelicolor, the ctc promoter was induced by salt shock, and the level of salt induction was significantly reduced in a sigH null mutant. The ctc promoter was also induced during the transition between exponential growth and stationary phase in S. coelicolor grown in liquid culture in the absence of exogenous stress. Unexpectedly, sigH was found to encode two N-terminally distinct isoforms, σ52 and σ37, which arise from translation initiation at two distinct in frame start codons (Viollier et al., submitted). Based on their apparent molecular weights and their ability to direct transcription of the ctc promoter, these two isoforms of σH almost certainly correspond to two sigma factors identified by Westpheling et al. (1985) in the original description of RNA polymerase heterogeneity in Streptomyces. The open reading frame (prsH) lying immediately upstream of sigH encodes a putative antisigma factor (PrsH stands for putative regulator of SigH), suggesting that σH may be regulated post-translationally (Viollier et al., submitted). Here, we show that transcription of the sigH operon is subject to temporal and spatial regulation during S. coelicolor colony development, and that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Transcription of sigH is developmentally regulated

Initial S1 nuclease mapping experiments showed that prsH and sigH form an operon transcribed from two promoters, initiating transcription 72 bp (sigHp1) and 133 bp (sigHp2) upstream of the prsH GTG start codon (Fig. 1). In B. subtilis, one of the two promoters of the sigB operon depends on σB itself (Yang et al., 1996). In the case of sigH, however, neither promoter resembles the ctc promoter that is transcribed by σH, and S1 nuclease protection analysis showed that both sigHp1 and sigHp2 were active in a constructed sigH null mutant (data not shown).

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Figure 1. Transcriptional organization of the prsH–sigH operon.

A. Genetic organization of the sigH locus showing the two N-terminally distinct isoforms of σH, σ52 and σ37, which arise from translation initiation at two distinct in frame start codons (Viollier et al., submitted). Initial S1 nuclease mapping experiments using probe A gave full-length protection, indicating that all sigH transcription initiated upstream of the PstI site internal to prsH.

B. Nucleotide sequence of the promoter region of the prsH–sigH operon showing the sigHp1 and sigHp2 transcription start points, their putative −10 and −35 sequences (underlined), the putative ribosome binding site (in bold) and the start of the prsH coding sequence.

C. High-resolution S1 nuclease mapping of the 5′ ends of the sigH transcripts using probe B. RNA was isolated from wild-type S. coelicolor A3(2) grown for 72 h on agar. Lanes labelled C, T, A and G represent a dideoxy sequencing ladder generated using the same radiolabelled oligonucleotide that was used to make S1 mapping probe B.

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Transcription of the sigH operon was monitored by S1 mapping during differentiation of wild-type S. coelicolor on solid medium and during growth in liquid culture (Fig. 2). Transcription from sigHp1 was readily detectable during all stages of liquid growth (Fig. 2A), but was seen only at the early time points on solid medium (Fig. 2A), suggesting that sigHp1 activity was associated with vegetative growth. In contrast, the sigHp2 transcript was undetectable during growth in liquid (apart from a faint signal in late stationary phase; Fig. 2A) and during vegetative growth in surface-grown cultures (Fig. 2B). However, on solid media, sigHp2 expression was strongly induced at the time of aerial hyphae formation and continued throughout sporulation (Fig. 2B). The time course of RNA samples used in this study was the same as that used by Kelemen et al. (1996) to analyse the transcription of sigF, which encodes a sigma factor required for spore maturation. sigF transcription is confined to spore compartments and is blocked by mutations that prevent sporulation septum formation (Kelemen et al., 1996; Sun et al., 1999). Comparison of the two sets of data showed that the appearance of the sigHp2 transcript precedes that of the sigF transcripts.

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Figure 2. A. S1 nuclease protection analysis of sigH and bldD transcription during growth of wild-type S. coelicolor in liquid medium [TSB containing 5% (w/v) PEG]. Mycelium was harvested at different time points during exponential growth (E), transition phase (T) and stationary phase (S).

B. S1 nuclease protection analysis of sigH and bldD transcription during development of wild-type S. coelicolor on solid medium (MM + mannitol). The time points (h) at which mycelium was harvested for RNA isolation and the presence of vegetative mycelium (V), aerial mycelium (A) and spores (S), as judged by microscopic examination, are shown.

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sigHp2 is active only in the aerial hyphae

The absence of sigHp2 transcripts in liquid culture (Fig. 2A), conditions that do not support differentiation of S. coelicolor, and the temporal correlation of sigHp2 promoter activity with the formation of aerial hyphae in surface-grown cultures (Fig. 2B) prompted us to examine the spatial location of sigHp2 activity within differentiating colonies. A sigHp2–EGFP transcriptional fusion was generated using the reporter plasmid pIJ8660 (Sun et al., 1999), which integrates site specifically into the S. coelicolor chromosome at the phage ΦC31 attB site. The resulting sigHp2–EGFP reporter plasmid, pIJ6910, was introduced into the morphologically wild-type S. coelicolor strain 916, and sigHp2 activity was monitored at different developmental stages using confocal fluorescence microscopy (Fig. 3).

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Figure 3. Localization of sigHp2 activity using the egfp reporter gene. Confocal microscope images of 916 (bldD+; morphologically wild type) and 1169 (bldD), both carrying sigHp2–egfp, are shown. Fluorescence images are on the right. Strains were inoculated adjacent to a coverslip inserted at an angle of ≈ 60° into an MM agar plate containing 0.5% (w/v) mannitol (a carbon source that permits sporulation of the bldD mutant). Cultures were incubated for 72 h, and images were taken of the aerial and vegetative mycelium attached to the coverslip.

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sigHp2-mediated green fluorescence was detected transiently between 44 h and 60 h and only in the aerial parts of the colonies (Fig. 3). Thus, sigHp2 activity not only correlated temporally with aerial growth, but was also spatially restricted to the aerial mycelium. Most of the fluorescence was localized to spore chains (Fig. 3), together with a few examples of apparently undifferentiated aerial hyphae. Most of the undifferentiated aerial hyphae and all the vegetative hyphae were non-fluorescent. The simplest interpretation of these results is that, in colonies of wild-type S. coelicolor, the sigHp2 promoter is primarily active in the spores. However, because of the long half-life of the EGFP protein (Corish and Tyler-Smith, 1999) and the results described below, it is possible that the sigHp2 promoter is activated just before sporulation septation.

Sporulation septum formation is not required for sigHp2 activity

The localization of sigHp2–EGFP fluorescence to spores raised the possibility that sigHp2 expression might depend on genes required for sporulation. To address this question, sigH transcription was monitored in time courses of RNA samples isolated from six sporulation mutants, whiA, B, G, H, I and J, grown on solid medium. These six ‘early’whi mutants are specifically affected in the initiation of sporulation, and three (whiA, B and G) are completely devoid of sporulation septa. Strong sigHp2 activity was detected in all six whi mutants, showing that P2 promoter activity does not depend on the formation of sporulation septa (data not shown).

sigH is expressed ectopically in a bldD mutant

To see whether transcription of the sigH operon was affected by any of the bld mutations that prevent the formation of aerial hyphae, RNA was isolated from bldA, B, C, D, G and H mutants grown on R5 solid medium. As these bld mutations exist in a complicated variety of genetic backgrounds (Table 1), only major variations in sigH mRNA levels with respect to wild-type S. coelicolor were considered potentially significant. The level of the transcript for hrdB, which encodes the principal, essential sigma factor of S. coelicolor, was monitored as an internal normalization control.

Table 1. Derivatives of S. coelicolor A3(2) used in this work.
StrainGenotypeReference
Wild typePgl+ SCP1+ SCP2+ Kieser et al. (2000)
916 hisA1 mthB2 pheA1 strA1 SCP1NF SCP2* Merrick (1976)
J1700 bldA39 hisA1 uraA1 strA1 SCP1 SCP2 Kieser et al. (2000)
J669 bldB43 mthB2 cysD18 agaA7 SCP1NF SCP2* Merrick (1976)
J660 bldC18 mthB2 cysD18 agaA7 SCP1NF SCP2* Merrick (1976)
1169 bldD53 hisA1 mthB2 pheA1 strA1 SCP1NF SCP2* Merrick (1976)
J774 bldD53 cysA15 pheA1 mthB2 strA1 SCP1NF SCP2* Merrick (1976)
WC103 bldG103 hisA1 uraA1 strA1 Pgl SCP1 SCP2 Champness (1988)
WC109 bldH109 hisA1 uraA1 strA1 Pgl SCP1 SCP2 Champness (1988)

Transcription from sigHp2 was detectable, but reduced, in five of the bld mutants, whereas transcription from sigHp1 seemed unaffected (Fig. 4A; data not shown). However, strikingly, sigHp2 transcription was dramatically upregulated in the bldD mutant J774 (Fig. 4A). To eliminate the possibility that differences in genetic background could account for this effect, sigH transcription was examined in RNA isolated from the original bldD isolate, 1169 (Merrick, 1976), and its congenic parent, 916; again, sigHp2 transcription was dramatically upregulated (data not shown). Moreover, in 1169 (bldD), sigHp2 transcripts were readily detectable in RNA isolated from liquid cultures under conditions in which no sigHp2 transcripts were detected in 916 (bldD+), indicating that repression of sigHp2 in liquid medium depends, directly or indirectly, on bldD. Although transcription from sigHp1 also appeared to be upregulated in J774 in Fig. 4A, in further independent experiments, sigHp1 upregulation was not observed consistently in the bldD background.

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Figure 4. A. S1 nuclease protection analysis of transcription of sigH and hrdB in representative mutants of four bld genes and in wild-type S. coelicolor. Mycelium was harvested for RNA isolation after 60 h growth on R5, a solid medium on which all the bld mutants fail to produce aerial mycelium, but on which the wild type sporulates normally.

B. Immunodetection of σH in crude extracts of 916 (bldD+) and 1169 (bldD). Crude extracts (10 µg) were prepared from 916 (lanes 1 and 2) and the bldD mutant (lanes 3 and 4) grown in J liquid medium (Puglia et al., 1995) for 12 h (lanes 1 and 3) and 36 h (lanes 2 and 4) and subjected to immunoanalysis using the σH antibody. The two primary translation products of σH37 and σ52) are indicated.

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Deregulation of sigHp2 in a bldD background was also shown through analysis of sigHp2–EGFP reporter plasmid, pIJ6910, in 1169 (bldD). Like many bld mutants, 1169 has a bald phenotype on R5 medium, but forms aerial hyphae and sporulates on minimal medium containing mannitol as carbon source (Merrick, 1976). Strong p2-mediated green fluorescence was detected in all tissues of a sporulating colony of 1169/pIJ6910 grown on minimal medium containing mannitol (Fig. 3). Ectopic expression of the sigHp2 promoter in the substrate mycelium was also detected when 1169/pIJ6910 was grown on R5 medium, when no aerial mycelium was formed (data not shown).

To see whether deregulation of the sigHp2 promoter correlated with overexpression of σH, mycelial extracts were prepared from liquid-grown cultures of 1169 (bldD) and 916 (bldD+). Immunoblot analysis of these extracts using σH polyclonal antiserum showed that both sigH primary translation products, σ52 and σ37, were present at higher abundance in the bldD mutant (Fig. 4B).

The sigHp2 promoter is a direct BldD regulatory target

Biochemical experiments were initiated to identify the regulatory protein(s) mediating the temporal and spatial control of sigHp2 activity. Gel retardation assays using crude mycelial extracts prepared from 916 (bldD+) detected an activity able to retard the migration of a 127 bp fragment carrying both sigH promoters (sigHp). Importantly, this binding activity was absent from the bldD mutant 1169 grown under the same conditions (data not shown). The sigHp-binding activity was subsequently purified from S. coelicolor 916 crude extracts using a procedure that relies primarily on a site-specific DNA affinity column as the final chromatographic step (Folcher et al., 2001). The sigHp-binding activity (as monitored by gel retardation) was partially purified by ammonium sulphate precipitation, DEAE–Sepharose chromatography and heparin–Sepharose chromatography, and then applied to a sigHp affinity matrix. After washing the column extensively, retained proteins were eluted stepwise with increasing concentrations of NaCl (0.2–1 M). These fractions were separated by SDS–PAGE and stained with Coomassie brilliant blue (Fig. 5A). A major band of 18 kDa correlated strongly with the gel-retarding activity; this protein, presumed to represent the sigHp-binding activity, was transferred to a polyvinylidene difluoride (PVDF)–nylon membrane, and its N-terminal sequence was determined by Edman degradation. The sequence obtained [S(S)EYAKQLGAKLRAIRTQQ] was identical to the predicted N-terminal sequence of BldD (Elliot et al., 1998), but lacking the N-terminal N-formylmethionine.

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Figure 5. A. The sigHp-binding activity in S. coelicolor 916 crude extracts was partially purified by sequential ammonium sulphate precipitation, DEAE–Sepharose chromatography and heparin–Sepharose chromatography. The active heparin–Sepharose fractions were applied to a sigHp affinity matrix, and proteins that interacted non-specifically were competed off the matrix using poly-(dI–dC)·poly-(dI–dC) (lane 1). Retained proteins were then eluted using increasing concentrations of NaCl in loading buffer (200 mM, lane 2; 400 mM, lane 3; 600 mM, lane 4; 800 mM, lane 5; and 1 M, lane 6). Samples (80 µl) of each eluted fraction (1ml) were resolved on a 15% (w/v) SDS–polyacrylamide gel and stained with Coomassie brilliant blue. The arrow on the right indicates BldD. The mobilities and molecular weights of the protein standards are shown on the left.

B. A 127 bp fragment carrying both sigH promoters (lane 1) was 32P labelled and incubated with BldD purified from either S. coelicolor (lane 2) or E. coli (lane 3), and DNA–protein complexes were separated from unbound DNA on a TBE–polyacrylamide gel.

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To confirm that BldD bound sigHp, the bldD gene was cloned in an Escherichia coli expression plasmid (pET11c) to generate pVHP364. sigHp-binding activity, not present in the vector-containing host [BL21(DE3)/pET11c; data not shown], was detected in crude extracts of BL21(DE3)/pVHP364 after induction with IPTG. When the recombinant BldD was purified from E. coli (as described for S. coelicolor), it retarded the 127 bp fragment in a manner indistinguishable from native BldD (Fig. 5B).

To localize the BldD binding site, the sigH promoter region was subcloned to provide fragments corresponding to p1(Fig. 1, co-ordinates 64–138) or p2(Fig. 1, co-ordinates 9–78 or 1–106). BldD only retarded the sigHp2 fragments, placing its binding site between nucleotide co-ordinates 9 and 78. The site was mapped more precisely by DNase I footprinting of the template strand (Fig. 6A); BldD protected a 44 bp sequence extending from −34 to +10 with respect to the sigHp2 transcriptional start site (Fig. 6B). The BldD footprints on the bldDp (Elliot and Leskiw, 1999) and sigHPp2 promoters are compared in Fig. 6B. Both footprints are of a similar size (42–44 bp), extending approximately from the −35 hexamer to about 10 bp downstream of the transcription start site. However, comparison of the sequences within these two footprints does not suggest a clear-cut consensus sequence for BldD binding (Fig. 6B).

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Figure 6. Localization of the BldD binding site by DNase I footprint analysis.

A. A fragment carrying the sigHp2 promoter (co-ordinates 1–106; Fig. 1B) was end labelled on the template strand, incubated alone (lane 1) or with BldD (50 pmol, lanes 2 and 3) purified from S. coelicolor(Fig. 5A) and then treated with DNase I for 5 (lanes 1 and 3) or 10 (lane 2) min (see Experimental procedures).

B. Comparison of the BldD footprints on the bldD (Elliot and Leskiw, 1999) and sigHp2 promoters. The extent of the DNase I-protected regions are highlighted, transcription start points are marked by arrows, and putative −10 and −35 promoter hexamers are underlined or overlined.

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The sigHp2 and bldD promoters are differentially regulated

Like sigHp2, the bldD promoter is repressed directly by the BldD protein (Elliot and Leskiw, 1999). However, the expression patterns of these two promoters are different. Although sigHp2 is active only in the aerial mycelium of wild-type S. coelicolor(Figs 2 and 3A), the bldD promoter is active during vegetative growth (Elliot et al., 1998). To confirm that these apparent differences are real and did not arise from differences in growth conditions or genetic background, bldD transcription was monitored in the same RNA samples that were used to monitor sigH expression during development in both solid and liquid grown cultures (Fig. 2). Although sigHp2 transcripts were detected only during aerial mycelium formation in surface-grown cultures of wild-type S. coelicolor and were hardly detectable in liquid culture, bldD transcription was readily detectable in all stages of liquid and surface-grown cultures.

Heat shock and ethanol also induce sigHp2 activity

The implication that σH is involved in the osmotic shock response (Viollier et al., submitted) prompted us to analyse the response of the sigH promoters to other stresses [Fig. 7; while this work was in progress, similar analyses were reported by Kormanec et al. (2000)]. Although sigHp1 was not affected by any of the stresses applied, sigHp2 was strongly induced by heat shock and, to a lesser extent, by ethanol, but not by osmotic or oxidative stresses. To see whether these effects on the sigHp2 promoter might be mediated through the expression of bldD, bldD transcription was monitored in the same RNA samples. bldD transcription was not affected by any of the applied stresses (Fig. 7). Given that BldD represses its own promoter (Elliot et al., 1998; Elliot and Leskiw, 1999) as well as sigHp2, if BldD were inactivated by heat or ethanol, bldD transcription might also be expected to be affected by these stresses. It is more likely, therefore, that the stress induction of sigHp2 is mediated by other, as yet unidentified, regulatory protein(s).

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Figure 7. S1 nuclease protection analysis of the effects of different stress conditions on transcription of sigH and bldD. Wild-type S. coelicolor was grown in liquid MM containing 5% PEG to late exponential phase and exposed to 0.5 M NaCl (lane 2), 5% (v/v) ethanol (lane 3), 1 mM H2O2 (lane 4) or temperature upshift to 41°C (lane 5) for 20 min. As a control, a sample was left untreated for 20 min (lane 1).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

sigH transcription is developmentally regulated

The activities of the two sigH promoters are strictly correlated with morphological development; the formation of aerial hyphae is accompanied by the strong induction of sigHp2 and the disappearance of sigHp1 transcripts. In addition, sigHp2 transcripts are virtually undetectable in liquid culture, conditions in which S. coelicolor does not differentiate. Using a sigHp2–EGFP transcriptional fusion, sigHp2-mediated fluorescence was found to be absent from the substrate mycelium, but was detected in spore chains and in a few aerial hyphae that were not overtly septating (without specific staining, septation can only be inferred indirectly, as the spore compartments begin to round off). However, monitoring transcription from sigHp2 in early whi mutants showed that the activation of sigHp2 does not depend on the formation of sporulation septa. These observations suggest that the developmental activation of sigHp2 might be associated with an earlier event, such as the cessation of growth of the aerial hyphae, rather than the induction of sporulation per se.

BldD is a regulator of the spatial organization of gene expression

bldD is one of 13 known loci required for aerial hyphae formation in S. coelicolor. On rich media, there is evidence for the involvement of a complex signalling cascade in the initiation of aerial mycelium formation, involving at least five different extracellular signals (Willey et al., 1993; Kelemen and Buttner, 1998). The steps in the signalling pathway have been defined genetically, and bldD is required to sense or respond to the last extracellular signal in the cascade (Willey et al., 1993). BldD acts as a repressor of its own promoter, bldDp (Elliot et al., 1998; Elliot and Leskiw, 1999), as well as sigHp2. Both promoters are strongly upregulated in a bldD mutant and, in both cases, BldD protects regions of similar sizes (42–44 bp) extending approximately from the −35 hexamer to about 10 bp downstream of the transcription start site (Fig. 6; Elliot et al., 1998; Elliot and Leskiw, 1999).

Recently, more targets for direct BldD repression have been identified (Elliot et al., 2001; J. Tenor, unpublished), including bldN, which encodes a sigma factor required for the initiation of aerial mycelium formation (Bibb et al., 2000), and whiG, which encodes a sigma factor that plays a critical role in triggering sporulation in aerial hyphae (Chater et al., 1989). Here, we show that BldD regulates tissue-specific expression of sigH; the sigHp2 promoter is repressed by BldD in vegetative hyphae, but this repression is released in sporulating aerial hyphae. Given the involvement of BldD in regulating these three key sigma factor genes, it seems clear that bldD is a critical regulator of biological processes associated with development, acting as a repressor of developmental genes during vegetative growth.

How is BldD activity regulated?

Our data show that BldD repression of sigHp2 is relieved at a certain point in the development of aerial hyphae. How is this relief of repression achieved? Many explanations are possible, including modification or proteolysis of BldD. In this regard, it is interesting to note that the Clp ATP-dependent protease plays an essential role in aerial mycelium formation in Streptomyces (de Crécy-Lagard et al., 1999). Analysis of the BldD amino acid sequence may yield important clues for other models. Although simple blast searches did not identify significant BldD homologues (Elliot et al., 1998), psiblast and pfam searches indicate that the N-terminal 69 amino acid residues of BldD show statistically significant similarity to the HTH-3 family of DNA-binding domains (pfam 01381; E-value 1.6 × 10−11). Members of the HTH-3 family include SinR, which acts as a tetrameric repressor that inhibits the expression of genes essential for entry into sporulation in B. subtilis, including spo0A and spoIIA (Mandic-Mulec et al., 1995). Thus, both BldD and SinR seem to act during vegetative growth to repress genes involved in development. The repressor activity of SinR is abolished through interaction with a partner protein, SinI, which disrupts the SinR tetramer through the formation of a SinI–SinR heterodimer (Lewis et al., 1998). BldD might be inactivated similarly through an interaction with a cognate partner protein. Finally, the only known bldD mutant allele (bldD53) carries a Tyr to Cys mutation at residue 62 (close to the C-terminal end of the proposed HTH-3-related DNA-binding domain), which led Elliot et al. (1998) to speculate that BldD might be regulated by phosphorylation.

The regulatory pathways controlling morphogenesis and stress responses in S. coelicolor are connected

The results reported here suggest that stress and development may be intimately connected in S. coelicolor. sigH was initially studied because of its involvement in stress responses. The smaller form of σH, σ37, was purified by virtue of its ability to direct in vitro transcription of a heterologous promoter, ctc (Westpheling et al., 1985; Viollier et al., submitted), which is part of the σB general stress response regulon in B. subtilis (Price, 2000). When introduced into S. coelicolor, the ctc promoter is induced by salt shock, and the level of induction is significantly reduced in a sigH null mutant (Viollier et al., submitted). In addition, sigHp2 is substantially induced by both heat shock and ethanol. However, it is now clear that sigH expression is also subject to developmental regulation, mediated by bldD, a gene forming part of the developmental cascade leading to the morphogenesis of a new cell type, the aerial hyphae.

The discovery that BldD spatially and temporally controls the p2 promoter of this stress-inducible gene (sigH) has two important implications. First, it identifies BldD as a regulator of tissue-specific gene expression during Streptomyces development and suggests that the restriction of sigHp2 activity to a specific stage and region during morphogenesis is achieved by modulating BldD activity in a tissue-specific manner. Secondly, it serves as a paradigm for linkage between stress response and developmental programmes. It seems unlikely that the connection between sigH expression and colony morphogenesis will prove to be unique. Instead, it suggests a more general regulatory organization, in which tissues with different developmental fates may have specialized stress response systems, reminiscent of the distinct heat shock stimulons induced during different physiological phases in liquid culture (Puglia et al., 1995).

Although it is clear that morphological development influences the expression of sigH, these results also raise the question as to whether stress response systems might influence developmental functions. Our recent findings suggest that the prsH sigH operon can influence development, perhaps through cross-talk with other sigma factors; although a sigH mutant sporulates normally, a prsH sigH double mutant is conditionally bald (P. H. Viollier, unpublished data), implying that PrsH must have a role beyond the regulation of σH. PrsH is homologous to SpoIIAB and RsbW, antisigma factors that regulate σF and σB, respectively, in B. subtilis (Alper et al., 1996; Yang et al., 1996), and PrsH probably plays a similar role as it co-purifies with σH (P. H. Viollier and A. Weihofen, unpublished data). However, unlike B. subtilis, the S. coelicolor genome encodes at least eight σB-type proteins. Two of the corresponding genes (sigI and sigJ) are adjacent to putative antisigma factor and anti-antisigma factor genes, three (sigH, sigL and sigM) are adjacent only to an antisigma factor gene, and three (sigG, sigK and sigN) are adjacent to neither. This genetic organization raises the possibility of cross-talk between the products of these loci, giving the potential for a complex stress response system that could be integrated into the network of developmental genes. Together with previous results (Vohradsky et al., 2000), the data presented here support the concept that evolutionary pressures have caused stress-regulatory systems to become adopted as part of a developmental programme in S. coelicolor.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains, plasmids, growth conditions and conjugal plasmid transfer from E. coli to Streptomyces

The S. coelicolor strains used are shown in Table 1. For RNA isolation from liquid cultures, tryptic soy broth (TSB) containing 5% PEG8000 (Sigma) was inoculated with spores of wild-type S. coelicolor A3(2), and mycelium were collected at different stages of growth. For RNA isolation from solid cultures, spores of S. coelicolor A3(2) or mycelial fragments of the whi and bld mutants were inoculated onto sterile cellophane disks on the agar surface of R5 (Kieser et al., 2000) or minimal medium (MM; Kieser et al., 2000) containing 0.5% (w/v) mannitol as carbon source and appropriate auxotrophic supplements. Unmethylated plasmids were conjugated from the dam dcm hsdS E. coli strain ET12567 (MacNeil et al., 1992) into S. coelicolor as described by Ryding et al. (1999).

RNA isolation and S1 nuclease mapping

RNA was isolated and S1 nuclease mapping was performed using 30 µg of RNA as described by Kelemen et al. (1996). All the probes were uniquely labelled on single 5′ ends. sigH probe A (Fig. 1A) was a BssHII–HindIII fragment (Fig. 1A) uniquely labelled at the BssHII site; the HindIII site was derived from the polylinker of pUC18. The remaining probes were generated by polymerase chain reaction (PCR) using pairs of cold and radiolabelled oligonucleotides. Oligonucleotides were radiolabelled on their 5′ ends using [γ-32P]-ATP (3000 Ci mmol−1) and T4 polynucleotide kinase. The oligonucleotides used were: for sigH probe B (Fig. 1A), 5′-CTAGCCACTGTGCGTGTTCGCGTG-3′ and 5′-GCAGCACCGACAGGTAGGCACC-3′ (labelled); for bldD, 5′-ACCTTAAAGGACGTCGGATATGCG-3′ and 5′-CGAGCTGTTTGGCGTATTCGCTGG-3′ (labelled); and for hrdB, 5′-CGGCCGCAAGGTACGAGTTGATGA-3′ and 5′-GCCATGACAGAGACGGACTCGGCG-3′ (labelled). The sequencing ladders were generated by dideoxy chain termination using the same radiolabelled oligonucleotide primer that was used to generate sigH probe B.

GFP fusions and monitoring GFP expression

A 314 bp DNA fragment carrying the sigHp2 promoter was amplified by PCR using the oligonucleotide primers 5′-ACCCCGGTACCGTGCTGTCACTCATAGTAAACACG-3′ and 5′-GGCACGGATCCGGTGCAGCTGAGGTTGTGTCTCG-3′ and chromosomal DNA as the template. The PCR product was cleaved with BamHI and KpnI and cloned into pIJ8660 (Sun et al., 1999) that had been cut with the same enzymes to generate pIJ6910; the insert was confirmed by sequencing. pIJ6910 and the parent vector, pIJ8660, were introduced into S. coelicolor 916 (bldD+) and 1169 (bldD) by conjugation from E. coli. Confocal microscopy was carried out using a Leica TCS NT confocal microscope (488 nm argon laser, × 63 objective, 0.5 numerical aperture dry PL Fluotar).

Purification of BldD from S. coelicolor

BldD was purified from S. coelicolor crude extracts using gel retardation as an assay. Unless otherwise stated, all purification steps were performed at 4°C. S. coelicolor 916 mycelium (200 g wet weight) grown to early exponential phase in a fermentor containing 50 l of J medium (Puglia et al., 1995) was lysed by sonication in 600 ml of TA buffer containing 50 mM NaCl and CompleteTM protease inhibitors (Roche Biochemicals). The cellular debris was removed by centrifugation for 30 min at 15 000 r.p.m. in a GSA rotor (Sorvall). The supernatant was clarified further by ultracentrifugation for 45 min at 45 000 g. Proteins that precipitated in 30% (w/v) ammonium sulphate were removed by centrifugation, and the ammonium sulphate concentration of the supernatant was brought to 60% (w/v). Precipitated proteins were collected by centrifugation and resuspended in 400 ml of TA buffer [10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1% (v/v) Triton X-100, 10% glycerol] supplemented with 50 mM NaCl. The sample was dialysed in TA buffer to give a conductivity equal to 50 mM NaCl. Two fractionation steps were performed with fast protein liquid chromatography (FPLC). First, the sample was loaded onto a 100 ml DEAE Fast Flow column (XK26/50; Amersham Pharmacia) that had been equilibrated in buffer TA containing 50 mM NaCl. Retained proteins were eluted in 540 ml of TA buffer by applying a linear NaCl gradient from 50 mM to 600 mM. Fractions containing sigHp-binding activity were pooled and diluted in TA buffer to give a conductivity equal to 100 mM. This sample was then applied to a 25 ml heparin Cl-6B Sepharose column (XK16/20; Amersham Pharmacia) that had been equilibrated in buffer TA containing 50 mM NaCl. After extensive washing, bound proteins were eluted with a 500 ml linear gradient ranging from 50 mM to 600 mM NaCl in buffer TA. Fractions that contained activity were dialysed and concentrated by ultrafiltration (YM10; Amicon) to 15 ml in buffer TA supplemented with 50 mM NaCl. Subsequently, affinity chromatography was performed using the DNA affinity column containing the sigH upstream region (described below). Immediately after incubation of the partially purified sample with the DNA affinity column, proteins that bound unspecifically were removed by performing a wash in the loading buffer (TA buffer containing 50 mM NaCl) that contained 0.2 mg ml−1 competitor DNA [poly-(dI–dC)·poly-(dI–dC)]. Thereafter, bound proteins were eluted by washing the magnetic beads stepwise in TA buffer with increasing NaCl concentrations. After each washing step, the NaCl concentration was raised by 200 mM until a final NaCl concentration of 1 M was reached. The 1 M NaCl wash was performed twice to maximize recovery of specifically bound protein. Three such elution cycles were performed until no gel-retarding activity remained in the flowthrough.

Preparation of a sigHp DNA affinity column

pVHP378 is a derivative of the vector pJS14 carrying a 127 bp fragment of DNA (5′-TTCCG ATTTGGGCCC……….TCACCGCTGAACCAC-3′) that contains both the sigH promoters (sigHp). Approximately 2 mg of a 300 bp biotinylated DNA fragment including this 127 bp sequence was prepared by setting up 200 PCR reactions using universal primer, biotinylated reverse primer and pVHP378 as the template. Unincorporated biotinylated primer was separated from the biotinylated PCR product by perfusion chromatography on a Poros HQ/F (Roche Biochemicals). The biotinylated sigHp fragment was coupled to streptavidin-coated magnetic particles (Roche Biochemicals) as described by Folcher et al. (2001). Briefly, 1 ml of streptavidin-coated magnetic particles was incubated with 4 ml of the sigHp fragment (0.5 mg ml−1) in TA buffer for 1 h with gentle agitation. The magnetic particles were washed three times in TA buffer containing 1 M NaCl and then resuspended in TA buffer containing 50 mM NaCl.

Expression of bldD in E. coli

The bldD gene was PCR amplified, cloned as an NdeI–EcoRI fragment into pET11c (Novagen) and its correct sequence confirmed. This plasmid, pVHP364, was used to overexpress bldD in E. coli BL21(DE3) (Novagen). An overnight culture of BL21(DE3)/pVHP364 grown at 37°C in LB was diluted 1:100 in LB, grown to an OD600 of 0.4 at room temperature and then induced with 1 mM IPTG. Cells were harvested after 4 h at room temperature. BldD was purified from E. coli as described for S. coelicolor.

DNase I footprinting

A fragment containing sigHp2 (co-ordinates 1–106; Fig. 1B) was PCR amplified using primers that introduced terminal HindIII and EcoRV sites and cloned into pGEMT easy (Promega) to create pJLT12. The fragment was released from pJLT12 by digestion with HindIII and EcoRV and end-labelled on the 3′ end of the template strand by filling in the HindIII site using the Klenow fragment of DNA polymerase (New England BioLabs) and [α-32P]-dATP. The radiolabelled probe (5 pmol) was incubated for 30 min at room temperature with ≈ 50 pmol of BldD (purified from S. coelicolor) in 20 µl of TA buffer containing 5 µg of poly-(dI–dC)·poly-(dI–dC) (Pharmacia). The DNA was partially digested with DNase I (Roche Biochemicals) at room temperature, precipitated and washed in ethanol, resuspended in 10 µl of stop solution (98% deionized formamide, 10 mM EDTA) and heat denatured (90°C for 5 min, then immediately placed on ice). Digestion products were resolved on an 8% (w/v) polyacrylamide gel and visualized by autoradiography.

Gel mobility shift assays

The probe used for the gel retardation assays was a 32P-labelled 127 bp fragment carrying both sigH promoters, isolated from pVHP385 (pVHP385 is a derivative of the vector pOK12 carrying a 127 bp BamHI–PmlI fragment). Protein samples were mixed with the labelled probe (2–5 ng) in a total volume of 20 µl of TA buffer containing 50 mM NaCl and 2 µg of competitor DNA [poly-(dI–dC)·poly-(dI–dC)] for 5 min at room temperature. The reaction mixtures were then resolved on a non-denaturing 5% (w/v) polyacrylamide gel in TBE buffer (90 mM Tris, 90 mM borate and 2 mM EDTA, pH 8) run at room temperature and constant voltage (7 V cm−1) for 2–4 h. After migration, gels were dried and the bands visualized by autoradiography.

Immunoblotting

Crude mycelial lysates containing 20 µg of protein were mixed with SDS–PAGE sample buffer, boiled, run on a 10% (w/v) SDS–polyacrylamide gel and transferred to PVDF membrane (Millipore) for immunodetection using the ECL Plus system (Amersham). Anti-σH antiserum (Viollier et al., submitted) was used as the primary antibody (1:10 000 dilution) and peroxidase-conjugated pig anti-rabbit IgG (Dako; 1:10000 dilution) as the secondary antibody.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Takeshi Murakami for N-terminal sequence analysis, and Rich Losick, Keith Chater, David Hopwood and Kien Nguyen for their comments on the manuscript. This work was supported by BBSRC grant 208/PRS12164 (to M.J.B.), by Swiss National Science Foundation grant 3100-059156.99/1 (to C.J.T.), by CNR grant AI99.0546.4 and a CNR short-term mobility grant (to L.M.), and by a grant-in-aid to the John Innes Centre from the BBSRC.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Footnotes
  1. Present addresses:

  2. School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

  3. Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5329, USA.

  4. §These authors contributed equally to this work.