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Abstract

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

A gene (sigB) encoding an alternative sigma factor σB in Streptomyces coelicolor A3(2) was isolated and characterized. It encodes a polypeptide of 281 amino acids (31 546 Da) and is highly homologous to Bacillus subtilisσB. The sigB coding region is preceded by four open reading frames (ORFs): dpsA, orfA, rsbB and rsbA in sequential order. RNA analyses revealed that rsbB, rsbA and sigB constitute an operon (sigB operon). Transcripts were produced constitutively from a promoter (sigBp2) upstream of the rsbB coding region, contributing to the basal level expression of σB protein. An inducible promoter (sigBp1) resembling the catB promoter (catBp) was located between the rsbA and sigB coding regions. Transcripts from sigBp1 dramatically increased as cells differentiated on solid media, at the stationary phase in liquid media or by osmotic stresses similar to the behaviour of catBp transcripts. Both catBp and sigBp1 promoters were recognized specifically by σB-containing RNA polymerase in vitro. Disruption of the sigB gene abolished not only the differentiation-associated expression but also the osmotic induction of the catB gene, indicating that catBp is under the control of σB. The sigB mutant exhibited a similar phenotype to the catB mutant, being sensitive to hyperosmolarity, blocked in forming aerial mycelium and with skewed antibiotic production. Therefore, we conclude that σB ensures the proper differentiation and osmoprotection of S. coelicolor cells, primarily via regulation of the expression of catalase B.


Introduction

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

In eubacteria, transcription initiation requires specificity proteins, known as sigma factors, that bind reversibly to the catalytically active core RNA polymerase (Burgess et al., 1969; Helmann and Chamberlin, 1988). Although all eubacterial RNA polymerases appear to share a conserved promoter specificity, imparted by the predominant sigma factor, recognition of diverse promoter sequences often requires a number of alternative sigma factors. From the complete genome sequences and by multiple sequence comparison of the conserved domains of sigma factors, the diversity of sigma factors can be estimated. For example, Escherichia coli, Mycobacterium tuberculosis and Bacillus subtilis are reported to contain seven, 13 and 18 different sigma factors respectively. Based on specific sequence patterns within the conserved domains, these sigma factors can be grouped further into several subfamilies, which are likely to represent some related functions in different bacteria. These include primary sigma, heat shock, extracytoplasmic function (ECF), flagella, B. subtilis sporulation or σB families. (Lonetto et al., 1994; Cho, 1999).

The σB (formerly σ37) of B. subtilis was originally discovered through its association with the RNA polymerase activity present in early stationary phase cells (Haldenwang and Losick, 1980). From many efforts to elucidate its functions, σB is now known to be the master regulator of the synthesis of stress-induced proteins as a functional homologue of E. coliσS (reviewed by Hengge-Aronis, 1996; Gaidenko and Price, 1998; Hecker and Völker, 1998). To investigate the general stress response of S. coelicolor at the molecular level, the existence of a functional homologue of B. subtilisσB or E. coliσS needs be examined.

In a pioneering work, Westpheling et al. (1985) reported that 37 kDa and 49 kDa proteins associated with Streptomyces coelicolor RNA polymerase are responsible for the specific transcription of the B. subtilis ctc (catabolite-controlled) promoter recognized by B. subtilisσB. They also showed that the promoter sequence of the endoH gene in Streptomyces plicatus, encoding an endoglycosidase H, is similar to the ctc promoter and is thus recognized by these proteins.

In a previous study, we found that the catB gene encoding a developmentally controlled catalase B is transcribed from a promoter closely resembling those recognized by B. subtilisσB, such as ctc and endoH promoters (Cho et al., 2000). We also found that catalase B is required for osmoprotection and proper differentiation of S. coelicolor. It contains a signal sequence similar to those of SapA and EndoH, which is cleaved off from the secreted protein upon differentiation. The observation that catB gene expression is elevated by osmotic stress and through differentiation, similar to the behaviour of katE, which is under the control of σB in B. subtilis (Engelmann et al., 1995), led us to postulate that there may indeed exist not only a structural but also a functional homologue of B. subtilisσB in S. coelicolor. Here, we describe the characterization of the sigB gene encoding an osmotic stress-responsive sigma factor σB that is also required for the onset of differentiation. We found that it is responsible for its own transcription and the regulation of catB gene expression. The role and regulation pattern of this new sigma factor is presented.

Results

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

Isolation of the sigB gene

In an attempt to identify novel sigma factors of B. subtilisσB type, responsible for stress response or differentiation in S. coelicolor, we designed polymerase chain reaction (PCR) primers from the conserved σB-type peptide motifs within regions 2.3/2.4 and 4.2 (Cho, 1999; Fig. 1). We obtained a single PCR product of the expected size, whose deduced amino acid sequence matched closely with B. subtilisσB-type sigma factors. This PCR product was used to screen the genomic phage library of S. coelicolor A3(2) M145. Three candidate clones all contained a common SmaI–BamHI fragment of 4.6 kb. The nucleotide sequence of this fragment was determined and examined for the presence of open reading frames (ORFs) using the frame program (Bibb et al., 1984).

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Figure 1. Comparison of S. coelicolorσB with related sigma factors. The predicted amino acid sequence of the sigB gene (ScoSigB) was compared with various σB-type sigma factors from B. subtilis (BsuSigB), Mycobacterium tuberculosis (MtuSigB), Staphylococcus aureus (SauSigB) and S. coelicolor (ScoSigF and ScoSigG). Fully conserved and similar residues are designated by asterisks, colons or dots, according to the definitions in the clustal w program (Thompson et al., 1994). The functional domains of sigma factors are shown in bold, according to Helmann and Chamberlin (1988). The arrows indicate the residues from which PCR primers were designed to amplify B. subtilisσB-type sigma factor(s) selectively as described in Experimental procedures.

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As shown in Fig. 2, the sequenced region contained five ORFs that were tentatively named dpsA, orfA, rsbB, rsbA, sigB and orfB. The initiation codon of rsbA overlaps the termination codon of rsbB (A TGA), suggesting that rsbB and rsbA may be co-transcribed and translationally coupled. A strong putative transcription termination signal was found between sigB and orfB.

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Figure 2. Restriction map and organization of the sigB operon. The structure of a 4611 bp SmaI–BamHI fragment containing the sigB gene is shown. The entire nucleotide sequence was deposited in the database under accession no. AF068267. The positions of restriction enzyme sites and the coding regions for dpsA, orfA, rsbB, rsbA, sigB and orfB are shown. S1probes (P1, P2) to map sigB transcripts are presented with asterisks at the radiolabelled 5′ ends. The protected fragments of P1(RT, T1) and P2probe (T2) are indicated by dotted lines. The transcription start sites (sigBp1 and sigBp2) are marked by bent arrows. The 344 bp AvaI–SalI fragment within the sigB coding region designated by a solid line represents the internal fragment used for sigB gene disruption. Abbreviations used for restriction enzymes are: A, AvaI; B, BamHI; Bc, BclI; C, ClaI; P, PvuII; Ps, PstI; S, SalI; Sm, SmaI; and St, StyI.

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Sequence comparison of ORFs with related proteins

The deduced amino acid sequence of each ORF was compared with related sequences in the database. The SigB protein was most similar to σF of S. coelicolor and showed significant similarity to σB of B. subtilis(Fig. 1). It shares about 45.1% identity (77.1% similarity) with S. coelicolorσF and about 32.7% identity (69.4% similarity) with B. subtilisσB. The C-terminal half of RsbA exhibits significant similarity to the kinase domain of anti-sigma factors RsbW and SpoIIAB of B. subtilis. The N-terminal half of RsbA does not show any similarity to known proteins. There exists a spacing of 128 bp between rsbA and sigB coding regions, unlike in the spoIIA and sigB operons of B. subtilis. RsbB does not share any significant sequence homology with the members of antagonist family proteins, such as RsbV, RsbS, RsbR and SpoIIAA of B. subtilis. Instead, it contains a weak transmembrane domain. DpsA contains conserved signatures of Dps family proteins.

The sigB gene is transcribed from two separate promoters

To define the transcription start sites for sigB expression, S1 nuclease mapping analyses were performed. Two different probes, one (P1) encompassing the sigB and rsbA intergenic region and the other (P2) encompassing the rsbA and rsbB upstream regions, were used (see Fig. 2). The P1 probe, consisting of 259 bp of intergenic region and 211 bp of vector DNA, was radiolabelled at +68 nucleotides (nt) downstream from the sigB initiation codon. It produced protected fragments of 121 and 259 nt (Fig. 2; see Fig. 3A). The 121 nt fragment is produced by a transcript (T1) initiated from a promoter (sigBp1) immediately upstream of the sigB coding region. The 259 nt fragment is produced by a transcript that is a readthrough (RT) from the rsbA coding region. The initiation site upstream of the rsbA gene was monitored by P2 probe, which was radiolabelled at +242 nt downstream from the rsbA initiation codon. The probe generated a protected fragment of 649 nt, demonstrating the presence of a second promoter (sigBp2) immediately upstream of the rsbB gene (Fig. 2; see Fig. 3B). These results suggest that sigB is transcribed as a monocistronic message from the sigBp1 and as a polycistronic message from the sigBp2 promoters. Inspection of sequences upstream of the sigBp2 start site revealed a −10 region with the sequence TAGGCT, similar to the consensus sequence recognized by the major σ factor of S. coelicolor, σHrdB (Buttner et al., 1990). There was, however, no recognizable consensus sequence in the −35 region. The sigBp1 promoter sequence resembled those recognized by B. subtilisσB (see below).

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Figure 3. Gene expression of sigB in vivo.

A. Changes in the level of sigB transcripts upon differentiation on solid medium. Total RNA was prepared from J1501 cells at the stages of substrate (S), aerial (A) and sporulated (Sp) mycelium on R2YE plates. S1 nuclease protection assays were performed with probe P1 (Fig. 2). RT indicates a readthrough transcript (259 nt), and T1 indicates the sigBp1 transcript (121 nt).

B. Changes in the level of sigB transcripts upon osmotic stress in liquid culture. J1501 cells were grown in YEME containing 10.3% sucrose for 15 (lanes 1 and 2) or 30 h (lanes 3 and 4) and subjected to 200 mM KCl treatment (lanes 2 and 4) or nothing (lanes 1 and 3) for 1 h before cell harvest. Total RNA was analysed by S1 nuclease mapping with P1 and P2 probes.

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Changes in the production of sigB transcripts in vivo

The changes in the amount of the two sigB transcripts, starting from the sigBp2 and sigBp1 promoters, were examined as S. coelicolor cells differentiated on solid plates. RNAs prepared from substrate (S), aerial (A) and sporulated (Sp) mycelium were analysed by S1 mapping with the P1 probe (Fig. 3A). The level of the sigBp2 transcript (RT) did not change significantly, whereas the sigBp1 transcript (T1) increased dramatically as cells differentiated. The effect of osmotic stress on the production of sigB transcripts was then examined in cells grown for 15 (at early exponential phase) or 30 h (at late exponential phase) in liquid culture (Fig. 3B). The amount of sigBp2 transcript (T2 or RT) monitored by probe 1 and 2 did not change regardless of the culture time or salt treatment. On the other hand, the sigBp1 transcript (T1) increased dramatically with salt treatment and upon prolonged culture. T1 transcript also increased with 4% NaCl and 34% sucrose, suggesting that it is increased by osmotic stress. It was not induced by ethanol (4%), heat (42°C) or H2O2 (0.1 mM).

σB recognizes both sigBp1 and catB promoters in vitro

The exact start site of the sigBp1 transcription was determined by high-resolution S1 mapping using probe P1 (Fig. 4A). The sequence of the proposed sigBp1 promoter is quite similar to that of catBp and other promoters known to be recognized by B. subtilisσB or σ3749 of S. coelicolor (Westpheling et al., 1985; Engelmann et al., 1995; see Fig. 4B). This implies that they may be recognized by S. coelicolorσB. To test this possibility, we examined in vitro transcription of catBp or sigBp1 promoter using S. coelicolor core RNA polymerase saturated with σB. His-tagged σB was overproduced and purified from E. coli. It was incubated with the purified RNA polymerase core enzyme (Kang et al., 1997) to prepare holoenzyme (EσB). As demonstrated in Fig. 4B, both catBp and sigBp1 promoters were transcribed in vitro by EσB. The sigBp1 promoter was recognized by EσB much better than catBp under the conditions used.

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Figure 4. Recognition of sigBp1 promoter by σB-containing RNA polymerase.

A. High-resolution S1 mapping of the sigBp1 start site. The RNA sample was prepared as in Fig. 3B at 15 h with (+) or without (–) 0.2 M KCl treatment. After S1 mapping with probe P1, protected DNA was loaded on a sequencing gel, with a sequencing ladder generated with the oligonucleotide labelled at the same 5′ end as probe P1. The initiation site was marked with an asterisk.

B. Comparison of the putative sigBp1 promoter with ctc of B. subtilis, catB and endoH promoters of Streptomyces spp. The conserved nucleotides within the −10 and −35 regions as well as transcription initiation sites are marked in bold. Those nucleotides shown to be important for ctc promoter activity in vivo and in vitro are underlined (Tatti and Moran, 1984; Ray et al., 1985).

C. In vitro transcription assay of sigBp1 and catB promoters by S. coelicolor RNA polymerase containing σB (EσB). The ratio of sigma factor to purified core RNA polymerase is indicated.

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The sigB mutant is impaired in differentiation and defence against osmotic stress

We constructed a sigB mutant from S. coelicolor A3(2) J1501 cells using an integration plasmid that contains an internal sigB gene fragment corresponding to amino acid residues 103–218 (see Fig. 2). Among several recombinants, whose expected genomic structure was verified by Southern analysis, one representative mutant (YD2108) was selected for further analyses. To our surprise, the sigB mutant appeared bald on an R2YE plate, unable to form aerial mycelium (Fig. 5). It also produced large amounts of actinorhodin precociously. The same phenotype was observed on NA but not SY plates. These phenotypes resembled that of the catB mutant very closely, except that the bald phenotype of the sigB mutant was slightly leakier than that of the catB mutant. To verify that the altered phenotype was indeed caused by the absence of the functional sigB gene, the 4611 bp SmaI–BamHI fragment containing the entire sigB operon was introduced into the sigB mutant by conjugal transfer as described in Experimental procedures. The exconjugant, which acquired the sigB gene in the chromosome via integration into the att site, resumed the wild-type colony morphology (Fig. 5).

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Figure 5. Morphological phenotype of the sigB mutant. Colony morphologies on R2YE plates of the wild type (upper left), catB mutant (upper right), sigB mutant (lower left) and the sigB mutant provided with a wild-type copy of the sigB gene (pSETsigB) by conjugal transfer (lower right) were observed under stereomicroscope. The apparent growth rate of the sigB mutant is almost the same as or slightly lower than that of the wild type on solid cultures. Both catB and sigB mutants appeared bald.

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We then examined the sensitivity of the sigB mutant to osmotic stress. As shown in Fig. 6A, the sigB mutant was not able to grow on NA plates containing 500 mM KCl, whereas the exconjugant cells that acquired the entire sigB operon (pSETsigB) grew well. When the mycelial growth rate of sigB mutant was measured quantitatively on NA plates containing different concentrations of KCl, we again observed that the sigB mutant was sensitive to salt and that osmoprotection was restored by introducing the wild-type sigB gene (Fig. 6B). The extent of osmosensitivity was similar between sigB and catB mutants.

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Figure 6. Salt sensitivity of the sigB mutant.

A. Osmosensitivity of the sigB mutant on solid medium. The sigB mutant harbouring the control pSET vector with (pSETsigB) or without (pSET152) the wild-type sigB gene was streaked on NA plates containing no additional or 500 mM KCl. Without a functional sigB gene, no growth was observed on a high-salt plate.

B. Osmosensitivity was assayed quantitatively by measuring the mycelial growth rates. The growth rates of catB mutant, sigB mutant and the complemented exconjugant (sigB + pSETsigB) were assessed by the rate of mycelial mass increase on NA plates containing various concentrations of KCl. The growth rate of untreated J1501 cells (doubling time of ≈ 8.7 h) was taken as 100.

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The antibiotic production of the sigB mutant was examined quantitatively by measuring the amount of actinorhodin (Act) and prodigiosins (Red) separately. Whereas the wild-type cells produced Red earlier than Act, the sigB mutant produced dramatically higher levels of Act precociously, while producing very low amounts of Red (Fig. 7A). The overproduction of Act pigment coincided with the higher and prolonged production of the actII-ORF4 transcript coding for the positive regulator of the Act biosynthetic genes (Fig. 7B). These behaviours of the sigB mutant in antibiotic production again paralleled the phenotype of the catB mutant (Cho et al., 2000). These results strongly suggest that the observed phenotypes of the sigB mutant might result primarily from the absence of catalase B, which is critically required for osmoprotection and proper differentiation of S. coelicolor cells (Cho et al., 2000).

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Figure 7. Antibiotic production in the sigB mutant.

A. The production of blue (Act; open and closed squares) and red pigment (Red; open and closed circles) in the wild type (empty symbols) and sigB mutant (filled symbols) grown on R2YE plates. The dotted vertical line marks the timing of aerial mycelium formation for the wild-type cells (≈ 60 h). To monitor Act production, A580 of pigments in 0.1 N NaOH solution from 10 mg (wet weight) of cells was measured. For Red production, A533 of the pigments in HCl–MeOH solution from about 33 mg (wet weight) of cells was measured.

B. The enhanced level of actII-ORF4 transcripts in the sigB mutant. Cells were grown for the indicated time on R2YE plates. In wild-type cells, aerial mycelium started to form after about 60 h. RNA was analysed by S1 mapping as described previously (Cho et al., 2000)

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Regulation of sigB and catB expression by σBin vivo

We then examined the expression of the sigB and catB genes in the sigB mutant under various conditions. As demonstrated in Fig. 8A, the osmotic induction of sigBp1 transcript was greatly mitigated in the sigB mutant, indicating that the induction of sigBp1 is mediated through σBin vivo as expected. The low level of induction remaining in the sigB mutant suggests that some σB-related sigma factor(s) of overlapping function may still act on the sigBp1 promoter to induce it partially. The osmotic induction of catB transcripts was abolished almost completely in the sigB mutant (Fig. 8B). This indicates that the induction of catB expression by high osmolarity is mediated almost entirely through σB. A low level of catB transcripts in the sigB mutant could have been produced by other σB-type sigma factors of overlapping recognition ability. The level of CatB protein paralleled that of transcripts monitored by Western analysis (Fig. 8C), suggesting that the osmotic induction of catB expression occurs mainly through transcriptional regulation. When the production of CatB protein was examined during growth on solid plates, we observed that the level of CatB did not increase nor was it processed in the sigB mutant upon prolonged culture (Fig. 8D). Therefore, the developmental regulation of catB expression and processing was critically affected by sigB mutation, as was its osmotic induction.

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Figure 8. Expression of the sigB and catB genes in the sigB mutant.

A. The sigB gene expression in both wild type and the sigB mutant was monitored by S1 mapping. Cells were grown in YEME containing 10.3% sucrose for 15 h and treated with (lanes 2 and 4) or without (lanes 1 and 3) 200 mM KCl for an additional 1 h.

B–D. catB gene expression was assessed by S1 mapping (B) and Western blot (C and D). To examine the salt induction of catalase B (CatB), cells were grown for 40 h on an NA plate containing no additional (lanes 1 and 3) or 200 mM KCl (lanes 2 and 4). To monitor the growth phase-dependent production of catalase B, cells were grown on R2YE plates, harvested at the indicated time and analysed by Western blot (D). The formation of aerial mycelium occurred at about 2.5 days in the wild-type cell. The precursor (92 kDa) and the processed (75 kDa) forms of catalase B are indicated by arrowheads.

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Discussion

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

σB-type sigma factors in S. coelicolor

The sequence similarity between the σBs of S. coelicolor and B. subtilis is particularly striking within regions 2.4 and 4.2 (Fig. 1). As the two regions are known to recognize the −10 and −35 elements of the promoters (Helmann and Chamberlin, 1988), both σB factors are predicted to recognize similar promoter sequences. The similarity of catBp and sigBp1 promoter sequences to those recognized by B. subtilisσB proves this prediction. In addition, both catBp and sigBp1 promoters are regulated in the same way, being induced at the stationary phase and upon differentiation and by osmotic stresses in a σB-dependent manner. Inspection of the ongoing S. coelicolor genome sequencing project (http://www.sanger.ac.uk/Projects/S_coelicolor) identified at least eight sigma factors of σB type, based on amino acid sequence homology. Among these, three sigma factors (σF, σG and σH) have been isolated and reported so far. It has been shown that σF recognizes the ctc promoter of B. subtilis (Kelemen et al., 1998) and functions at the late stage of sporulation, being necessary to form spores from the aerial mycelium (Potúčkováet al., 1995; Kelemen et al., 1996, 1998). The functional role of σG has not been suggested yet, as the disruption of its gene does not affect either differentiation or stress response (Kormanec et al., 1999). The gene for σH (sigH) has been identified and shown to be induced by osmotic stress and heat shock using separate promoters (Kormanec et al., 2000). σB is unique in that: (i) its gene expression is induced by osmotic stress and through the differentiation process using a common promoter; (ii) it functions in protecting S. coelicolor cells against osmotic stress and in proper morphological and physiological differentiation. The latter point distinguishes it from its counterpart in B. subtilis, which is known to be unnecessary for spore formation (Igo et al., 1987).

Regulation of σB activity

The σB activity of B. subtilis is controlled by a multicomponent network, which responds to a variety of environmental and metabolic stresses. All the signals appear to control σB activity by altering the ability of the anti-σB factor, RsbW, to bind and inactivate σB (Benson and Haldenwang, 1993). We postulate that, in the S. coelicolor sigB operon, the rsbA gene encodes a putative anti-σB factor, based on its sequence similarity to RsbW. There is preliminary evidence that RsbA interacts with σBin vitro (Cho, 1999). There is no RsbV-like protein within the sigB operon in S. coelicolor. Instead, a putative antianti-sigma function of RsbV type could be postulated for the unlinked putative antianti-sigma factor such as BldG (Bignell et al., 2000). Further investigation is needed to find out the mechanism that controls σB activity. At this point, it is worth emphasizing the unique operon structure of sigB in S. coelicolor; especially the presence of an internal promoter from which only the monocistronic mRNA for σB is transcribed. This operon structure is different from any of the known σB-type operons, such as B. subtilis sigB and sigF (Kalman et al., 1990), Staphylococcus aureus sigB (Wu et al., 1996) and S. coelicolor sigH (Kormanec et al., 2000), in which the sigma genes are always co-transcribed with the upstream anti-sigma factor (-like) genes.

The induction of the monocistronic sigB transcript upon osmotic stress and through the differentiation process seems to be responsible for the increased synthesis of σB protein under these conditions. The amount of σB protein in S. coelicolor in rapidly growing as well as in stationary phase cells is not high enough to be detected clearly by Western blot. Only in the salt-induced cells were we able to detect a significant amount of σB protein (data not shown). Therefore, the increase in transcripts, at least under osmotic stress conditions, leads to the increase in σB protein level. As the inducible sigBp1 promoter is recognized by σB itself, the initial triggering for salt- and growth phase-specific induction of sigBp1 must come from enhancement of the already existing activity of σB by these environmental cues. After that, the increased production of σB forms a positive feedback loop amplifying the signal. The ways by which the initial triggering of sigBp1 induction happens as well as the existence of a negative feedback loop need to be elucidated in future.

σB is involved in the differentiation and osmotic stress response, mainly by regulating the production of catalase B

The resemblance of the sigB mutant phenotype to that of the catB mutant and the observation that the catB gene is transcribed by σB-containing RNA polymerase in vivo and in vitro suggest that σB exerts its effect on osmotic response and differentiation, mainly via inducing catalase B. Extracellular complementation between the sigB and catB mutants also suggests that they can be grouped together, catB being epistatic to sigB (data not shown). Not only the scarce production but also the lack of any processed form of catalase B in the sigB mutant upon long-term culture suggest that σB may govern catB gene expression at both the transcriptional and the post-translational levels. Even though a strong correlation between the increased production and processing of catalase B and differentiation has been observed, how and why they are correlated is not understood. On prolonged incubation, the sigB mutant starts to form aerial mycelium from the edge of the colonies. The differentiated (leaky) sigB mutant cells were found to produce some catalase B, suggesting that the catB gene might be transcribed by yet another σ factor that becomes available upon long-term culture (data not shown). Currently, our observations strongly suggest that σB is in charge of catB gene expression in vivo, and thus governs the osmotic response and differentiation process of S. coelicolor. However, this postulate does not exclude the possibility that σB may control the expression of genes other than catB required for the onset of differentiation and osmoprotection. In order to understand the role of σB in S. coelicolor, not only the mechanism of sensing the environment to increase σB activity but also the spectrum of its downstream target genes need be elucidated.

Experimental procedures

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

Bacterial strains and culture conditions

Growth and maintenance of S. coelicolor A3(2) strains (J1501, M145 and their derivatives) were performed as described by Hopwood et al. (1985) and Cho et al. (2000). For liquid culture, YEME medium containing either 34% or 10.3% sucrose was inoculated with pregerminated spores (about 108≈109 spores 100 ml broth). For plate culture, 107 pregerminated spores or patches of mycelia were streaked on R2YE, NA or minimal agar media. To facilitate harvesting of aerial and sporulated mycelia, inocula were spread on cellophane membrane on solid media. The growth rates and phases were determined as described by Cho and Roe (1997). To apply osmotic stress in liquid culture, 200 mM KCl was added to cultures of exponentially growing cells in YEME for 1 h before harvest.

Preparation of cell extracts and determination of antibiotic pigments

Harvested mycelia were suspended in TGED [50 mM NaCl, 50 mM Tris-HCl, pH 7.8, 5% glycerol, 0.1 mM EDTA, 0.1 mM dithiothreitol (DTT)] buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF). They were disrupted by sonication (Sonics and Materials), and the suspension was clarified by centrifugation at 4°C. The concentration of total protein in the cell extract was determined using a protein assay kit (Bio-Rad). Extraction of antibiotic pigments and their spectrophotometric quantification were carried out as described by Hobbs et al. (1990) and Adamidis et al. (1990).

DNA manipulations

Restriction and modifying enzymes were used according to the manufacturer's recommendations (Poscochem, Boeringer Mannheim, NEB). Standard methods for DNA and phages were used as described by Sambrook et al. (1989). DNA fragments were purified from agarose gels using GeneClean kit II (BIO101) or the freeze-squeeze method. E. coli DH5α, methylation-negative E. coli ET12567 (MacNeil, 1988) and S. coelicolor A3(2) J1501 cells were used as hosts for various recombinant DNAs.

Library screening to isolate the sigB gene

Oligonucleotide primers for PCR were designed from the conserved amino acids among B. subtilisσB-type sequences considering the biased codon usage of S. coelicolor. The forward primer of 24 nucleotides (5′-GCS RTS CCS AYS RTC RTS GGS GAG-3′) and the backward primers of 23 nucleotides (5′-GAG AYS YRS RHC TGS GAS AYS CC-3′) were used. The PCR-amplified fragment was verified by sequencing and used as a probe to screen the λEMBL3 genomic library of S. coelicolor A3(2) M145. Among 20 primary phage candidates, λ12, λ13 and λ16 contained a common region hybridized by the PCR product. The nucleotide sequence of the 4611 bp SmaI–BamHI fragment encompassing the whole ORFs for OrfB, σB, RsbA, RsbB, OrfA and DpsA was determined and deposited in the database under accession number AF068267.

Expression and purification of proteins

The sigB coding region was amplified by PCR with a mutagenic forward primer containing the start codon and a backward primer containing the BamHI site immediately downstream of the termination codon (Cho, 1999). The resulting PCR product of ≈ 900 bp, digested with NdeI and BamHI, was cloned in pET-15b (Novagen) to yield pET15209. A fresh colony of E. coli harbouring pET15209 was grown and treated as described previously (Cho et al., 2000). The recombinant His-tagged σB protein was recovered from the inclusion body after solubilization with 7.5 M guanidine–HCl. The protein was allowed to renature by dialysing twice against the binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 0.5 M NaCl). The dialysate was subjected to chromatography through an Ni-NTA column (Novagen). The eluate by 1 M imidazole was concentrated to about 0.5 mg ml−1 and dialysed against storage buffer (50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 0.1 M KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol).

S1 nuclease mapping

The probes used for S1 nuclease mapping of the sigB transcripts are shown in Fig. 2. P1 probe was prepared from pUC203, a pUC18 derivative containing a 306 bp PCR product encompassing the intergenic region of rsbA and sigB. PCR amplification was carried out with the backward primer 5′-C GAA GAA CAG CTT CGA CAA CTC C-3′ (5′ position at +168 nt relative to the sigBp1 start site) and the forward primer 5′-GAT CTG GTG CGG GTT CAC ACG GAC-3′ (5′ position at −138 nt relative to the sigBp1 start site). pUC203 was digested with StyI located at position +121 from the transcription start site (+68 from the initiation codon), labelled at the 5′ phosphate of the StyI-cut end and then digested with PvuII to recover 470 bp labelled probe. P2 probe, which spans the upstream region of rsbA and rsbB, was generated by digesting a 4.6 kb SmaI–BamHI fragment partially with SmaI. The hybridization reaction was performed at 55≈65°C for over 8 h using 50 µg of RNA. For S1 mapping of catB and actII-ORF4 transcripts, 100 µg of RNA was hybridized with the radiolabelled DNA probes as described previously (Cho et al., 2000).

In vitro transcription assay

In vitro transcription assays were carried out as described by Kang et al. (1997) with slight modifications. Core RNA polymerase (1.5 pmol) was incubated with 10 pmol or more of His-tagged σB for 30 min at 37°C to produce holoenzymes. Template DNAs containing catBp or sigBp1 promoter (0.2 pmol) were added and incubated for an additional 30 min to allow sufficient formation of the promoter open complexes. At 30 s after the initiation of transcription by adding 0.1 mM each of NTP containing 32P-labelled CTP, heparin (50 µg ml−1) was added to restrict the transcription to a single cycle. Transcripts were resolved in 5% PAGE containing 7 M urea, followed by autoradiography.

Gene disruption and complementation

The 344 bp AvaI–SalI fragment, containing the internal sigB fragment corresponding to regions from 2.3 to 3 of σB (103rd to 218th residues), was cloned into pDH5 (Hillemann et al., 1991) to construct pDH2344. Alkaline-denatured pDH2344 DNA isolated from the non-methylating E. coli strain ET12567 was introduced into J1501 protoplasts by transformation. Single cross-over recombinants were identified as thiostrepton-resistant colonies, which were passaged to isolate single colonies. One representative recombinant (YD2108) was chosen for further analyses after verification of its gene structure by Southern analysis. For complementation experiments, the 4611 bp SmaI–BamHI fragment containing the entire sigB operon was isolated from pUC246 (Cho, 1999) and cloned into pSET152 (Bierman et al., 1992), resulting in pSETsigB. The introduction of pSETsigB into YD2108 by conjugation and the isolation of exconjugants were performed as described by Cho et al. (2000).

Acknowledgements

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

We are grateful to Drs G. Kelemen and M. Buttner for kindly sharing unpublished results, to J.-B. Bae for his technical assistance with protein purification, to M.-Y. Hahn for help with in vitro transcription assay, and to Y.-G. Nam for DNA sequencing. This work was supported by the Genetic Engineering Research Grant (1998) and Basic Science Research Grant (2000) from the Korean Ministry of Education. Y.-H. Cho was the recipient of a BK21 Postdoctoral Fellowship from the Korean Ministry of Education.

References

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