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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The chaplin and rodlin proteins together constitute the major components of the hydrophobic sheath that coats the aerial hyphae and spores in Streptomyces, and mutants lacking the chaplins are unable to erect aerial hyphae and differentiate on minimal media. We have gained insight into the developmental regulation of the chaplin (chp) and rodlin (rdl) genes by exploiting a new model species, Streptomyces venezuelae, which sporulates in liquid culture. Using microarrays, the chaplin and rodlin genes were found to be highly induced during submerged sporulation in a bldN-dependent manner. Using σBldN ChIP-chip, we show that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σBldN. sven3186 (here named rsbN for regulator of sigma BldN), the gene lying immediately downstream of bldN, was also identified as a target of σBldN. Disruption of rsbN causes precocious sporulation and biochemical experiments demonstrate that RsbN functions as a σBldN-specific anti-sigma factor.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Differentiation of the filamentous bacteria Streptomyces on solid medium requires the formation of specialized reproductive structures called aerial hyphae, which have to escape the aqueous environment of the vegetative mycelium and grow into the air. These aerial hyphae subsequently arrest growth and the multigenomic apical cell differentiates into long chains of pigmented, unigenomic spores (Elliot et al., 2008; Flärdh and Buttner, 2009; McCormick and Flärdh, 2012). Studies on the classical model species Streptomyces coelicolor show that, in order to break surface tension, streptomycetes have to coat their aerial hyphae in an extremely hydrophobic sheath that is absent from the vegetative hyphae growing in the aqueous phase, and, on rich media, they must also produce a surfactant peptide called SapB (Hopwood and Glauert, 1961; Wildermuth et al., 1971; Elliot and Talbot, 2004; Willey et al., 2006; Flärdh and Buttner, 2009). The hydrophobic sheath, which permits the aerial hyphae to escape surface tension and likely also prevents their desiccation in air, is made up of two families of proteins: the chaplins and the rodlins.

The chaplins are surface-active proteins that can be divided into two classes: the short chaplins and the long chaplins (Claessen et al., 2003; Elliot et al., 2003). All chaplins share a highly conserved, hydrophobic domain of ∼ 50 amino acids, termed the chaplin domain. The short chaplins consist of a single chaplin domain preceded by an N-terminal Sec secretion signal. The long chaplins have an N-terminal Sec secretion signal followed by two iterations of the chaplin domain separated by a flexible linker, then a long peptidoglycan-spanning domain and a C-terminal ‘sorting signal’ that targets them for covalent attachment to the cell wall peptidoglycan of the aerial hyphae and spores by sortase enzymes (Claessen et al., 2003; Elliot et al., 2003; Duong et al., 2012). The number of short and long chaplins varies from species to species; of the species most discussed in this report, Streptomyces venezuelae has two long chaplins (ChpB and ChpC) and four short chaplins (ChpE–H), whereas S. coelicolor has three long chaplins (ChpA–C) and five short chaplins (ChpD–H). ChpC, ChpE and ChpH are the only chaplins conserved in all Streptomyces species whose genome sequences are available. The chaplins self-assemble via their chaplin domains into amyloid-like filaments on the surface of the aerial hyphae and spores (Claessen et al., 2003; Elliot et al., 2003; Capstick et al., 2007; 2011; Di Berardo et al., 2008; Sawyer et al., 2011). It was originally thought likely that the short chaplins attached to the aerial surface through heteropolymerization with the cell wall-anchored long chaplins (Claessen et al., 2003; Elliot et al., 2003), but it is now clear that mutants lacking the long chaplins still assemble amyloid filaments of the short chaplins on their aerial surfaces (Duong et al., 2012).

The second family of proteins associated with the hydrophobic sheath is the rodlins (Claessen et al., 2002; 2004). S. venezuelae has three rodlins (RdlA–C) and S. coelicolor has two (RdlA and RdlB). The rodlin proteins are not required for aerial mycelium formation and mutants of S. coelicolor lacking the two rdl genes still form a hydrophobic sheath. However, instead of having the ‘basketwork of paired rodlets’ surface ultrastructure that is characteristic of the wild type when examined by scanning electron microscopy, the rdl null mutant exhibits a disordered network of fine filaments, suggesting that the chaplin filaments are organized into the larger ‘rodlet’ ultrastructure by the rodlins (Claessen et al., 2002; 2004).

Streptomyces coelicolor SapB is a hydrophobic, lantibiotic-like peptide specified by the rapid aerial mycelium formation (ram) gene cluster (Willey et al., 1991; Ma and Kendall, 1994; Kodani et al., 2004). SapB is the ribosomally encoded product of the ramS gene, which is translated as an inactive prepeptide that undergoes extensive post-translational modification before being cleaved to yield mature SapB (Kodani et al., 2004). On minimal media, SapB is not expressed and aerial development is exclusively dependent on the chaplins. However, on rich media, S. coelicolor has to produce SapB in addition to the chaplins to allow efficient formation of aerial hyphae (Willey et al., 1991). SapB is secreted during the vegetative phase of development, and available evidence suggests that it coats both the nascent aerial hyphae and the air–water interface to facilitate the emergence of aerial filaments into the atmosphere (Tillotson et al., 1998).

Expression of the chp genes is initiated prior to aerial mycelium formation on both rich and poor media, and a strain deleted for all eight chaplin genes cannot produce aerial hyphae on minimal media and is severely deficient on rich media (Claessen et al., 2003; Capstick et al., 2007). Strains that cannot make both the chaplins and SapB cannot produce aerial hyphae under any growth conditions (Capstick et al., 2007). When quantified in vitro, the chaplins and SapB both have powerful surfactant activity at air–water interfaces, and this ability is presumably central to their partially overlapping functions (Tillotson et al., 1998; Claessen et al., 2003; Capstick et al., 2007). In static liquid cultures, the chaplins also mediate attachment of S. coelicolor to surfaces via fimbriae composed of amyloid chaplin fibrils (de Jong et al., 2009).

bld mutants are a class of developmental mutants that cannot erect aerial hyphae and therefore appear ‘bald’, lacking the fuzzy morphology of the wild type (Flärdh and Buttner, 2009). One of the key Bld regulators of morphological differentiation in Streptomyces is an ECF sigma factor, known as σBldN in S. coelicolor and σAdsA in Streptomyces griseus (Bibb et al., 2000; Yamazaki et al., 2000). As the name implies, mutations in bldN block aerial mycelium formation. Until now, the only known direct biochemical target of σBldN was bldMp1, one of two promoters of bldM, a gene that encodes an atypical response regulator also required for aerial mycelium formation in S. coelicolor (Bibb et al., 2000; Molle and Buttner, 2000).

Despite the central importance of the hydrophobic sheath in Streptomyces development, no direct regulators of the chp and rdl genes have been identified. In S. coelicolor growing on solid medium, mutations in all the bld genes tested block chp gene expression (Elliot et al., 2003), implying that the formation of nascent aerial hyphae itself acts as a morphological checkpoint for chp gene expression, and that the nascent hyphae can sense they have left the aqueous environment (Claessen et al., 2006).

Significant progress is being made in understanding the cell biological processes underlying morphogenesis in Streptomyces (reviewed in Flärdh and Buttner, 2009), and all of the bld and whi developmental master regulators defined by classical mutant hunts have been cloned and characterized. However, what is critically needed is an understanding of the global regulatory networks that connect the developmental cell biological processes to these master regulators, but, to date, only the regulons of BldA, BldD and BldH (also called AdpA) have been characterized in depth (Yamazaki et al., 2000; 2003a,b; 2004; Elliot et al., 2001; Takano et al., 2003; Tomono et al., 2005a,b; Hirano et al., 2006; Chandra and Chater, 2008; Chater and Chandra, 2008; den Hengst et al., 2010; Higo et al., 2011; 2012). Characterizing the regulatory networks that govern morphological development using S. coelicolor as an experimental system poses certain logistical challenges. S. coelicolor sporulates only on solid medium and the differentiating part of the colony (the aerial mycelium) constitutes only ∼ 5% of the total biomass, making the application of global techniques like chromatin immunoprecipitation-microarray (ChIP-chip) analysis to development-specific transcription factors problematic. In this report we take advantage of S. venezuelae as a new model system for the genus (M. Bibb et al., unpublished). In contrast to S. coelicolor, S. venezuelae sporulates in liquid culture and > 90% total biomass is converted into spores (Glazebrook et al., 1990), allowing optimal application of global approaches to the analysis of cellular differentiation.

Here we show that expression of the chp and rdl genes is highly induced in S. venezuelae during sporulation in liquid culture and that this expression depends on the bldN gene. We show using ChIP-chip that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σBldN. Further, we identify rsbN (sven3186), the gene lying immediately downstream of bldN as another member of the σBldN regulon. Disruption of rsbN causes precocious sporulation, and biochemical experiments show that RsbN functions as a σBldN-specific anti-sigma factor.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The chaplin and rodlin genes are expressed during submerged sporulation in a bldN-dependent manner

Having established conditions in which > 90% S. venezuelae mycelium differentiated into spores in MYM liquid culture (Glazebrook et al., 1990; M. Bibb, unpublished), we wanted to know whether the components of the hydrophobic sheath were expressed under these conditions. To answer this question, DNA microarray experiments were performed to determine the transcriptional profile of the chp and rdl genes in wild-type S. venezuelae during submerged sporulation. RNA samples were prepared at 2 h intervals from 8 to 20 h, by which time sporulation was nearing completion, and, following cDNA synthesis and labelling, samples were hybridized to DNA microarrays. Analysis of the DNA microarray data from three independent experiments revealed that transcription of all of the chp and rdl genes was dramatically induced during submerged sporulation (Fig. 1), implying that expression of the chaplins and rodlins is ‘hard-wired’ into the sporulation programme, regardless of whether it occurs on plates or in liquid culture. In parallel with these experiments, to examine the effects of σBldN on genome-wide transcription, we isolated RNA samples at 2 h intervals from a liquid culture of a constructed bldN null mutant grown under identical conditions to the wild type. Analysis of the DNA microarray data from three independent experiments revealed that the chp and rdl genes were not induced in the bldN null mutant (Fig. 1).

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Figure 1. Transcriptional expression profiles of the chp and rdl genes in wild-type S. venezuelae during submerged sporulation and in a congenic bldN null mutant grown under identical conditions. In each panel, the x-axis indicates the age of the culture in hours, and the y-axis indicates the per gene normalized transcript abundance (log2). The identifiers corresponding to the gene names are: chpB (sven1616), chpC (sven1269), chpE (sven1437), chpF (sven4645), chpG (sven4651), chpH (sven1270), rdlA (sven4628), rdlB (sven4631) and rdlC (sven4632).

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The chaplin and rodlin genes are direct biochemical targets of σBldN

To determine whether chp and rdl gene expression was directly or indirectly dependent on σBldN, σBldN ChIP-chip experiments were performed. As described in Experimental procedures, an S. venezuelae rsbN null mutant (see below), grown under the same conditions used for the array-based transcriptional profiling experiments, was subjected to formaldehyde cross-linking, lysis and sonication after 13 h of growth, a time point at which σBldN expression was evident, as judged by immunoblotting (Fig. S1). After immunoprecipitation using a σBldN-specific polyclonal antibody, the DNA was labelled with Cy3 and hybridized together with a total DNA control, labelled with Cy5, to a high-density microarray representing the S. venezuelae genome. Three biological replicates were performed in the bldN+ strain and the values were averaged. Peaks in the fluorescence ratio indicate regions of the chromosome that are bound by σBldN (Fig. 2). As a control, a ChIP-chip experiment was performed using the bldN null mutant to eliminate any potential signals arising from cross-reaction of the antibody with other transcription factors (Fig. 2).

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Figure 2. σBldN ChIP-chip data for selected targets. The data for the bldN+ strain (blue dots) show averaged values from three independent biological replicates, whereas the data for the bldN null mutant control strain (green dots) are from a single experiment. DNA obtained from immunoprecipitation of σBldN was labelled with Cy3 and hybridized to DNA microarrays together with a total DNA control that was labelled with Cy5. The data are plotted as Cy3/Cy5 ratios (y-axis), as a function of chromosome location (x-axis). Plots span approximately 8 kb of DNA sequence. This figure is available in colour online at http://wileyonlinelibrary.com.

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Prior to these experiments, in work done in S. coelicolor, the p1 promoter of bldM was the only previously characterized σBldN target promoter (Bibb et al., 2000). Examination of the ChIP-chip data showed that the bldMp1 promoter is also a direct biochemical target of σBldN in S. venezuelae (Fig. 2; see also the bldMp1 in vitro transcription data described below). Strikingly, further examination of the ChIP-chip data showed that the promoter regions of all of the chp and rdl genes were bound by σBldNin vivo, implying they are also directly activated by σBldN (Fig. 2).

To extend the ChIP-chip analysis, the 5′ ends of all of the chp transcripts and the rdlA and rdlB transcripts were determined by S1 nuclease protection analysis (Fig. 3), using RNA isolated from a 16 h liquid culture of wild-type S. venezuelae, a time point at which the microarray transcriptional profiling showed peak expression of the chp and rdl genes (Fig. 1). As expected, in all the cases examined (chpC, chpE, chpH, rdlA, rdlB), no transcripts were detected in RNA isolated from an equivalent culture of the congenic bldN null mutant (Fig. 3). The promoter sequences of the chpB, chpE, chpF, chpG, chpH, rdlA and rdlB genes were aligned using their transcription starts (Fig. 4A). The putative −10 and −35 sequences of the rdlA, rdlB and chp promoters aligned well with each other, and also with those of the bldMp1 promoter (Fig. 4A), establishing a conserved motif for σBldN-dependent promoters, 5′-CGNAACC(N15–16)(C/A)GTT-3′. Although the 5′ end of the rdlC transcript was not determined experimentally, a strong match to this consensus sequence was identified upstream of rdlCFig. 4A), with the putative −10 sequence, AGTT, lying 66 bp upstream of the rdlC ATG start codon. A sequence logo for σBldN-dependent promoters, shown in Fig. 4B, was derived using the sequences shown in Fig. 4A as input for WebLogo (http://weblogo.berkeley.edu). In addition to the previously known S. coelicolorσBldN target promoter, bldMp1 (Bibb et al., 2000), the S. coelicolor chpE and chpH promoters have also been defined by S1 nuclease mapping (Elliot et al., 2003), and these promoters align well with the CGNAACC(N15–16)(C/A)GTT-3′ motif (Fig. S2), suggesting that chpE and chpH are also directly activated by σBldN in S. coelicolor.

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Figure 3. S1 nuclease mapping of the 5′ ends of the chp and rdl transcripts in RNA isolated from wild-type S. venezuelae (WT) or the congenic bldN null mutant (bldN). Lanes marked P indicate a sample of the undigested probe. The size standards (MW) were a heat-denatured, [γ-32P]-labelled HinfI digest of ΦX174 DNA.

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Figure 4. A. Sequence alignment of σBldN target promoters. For the chpB, chpE, chpF, chpG, chpH, rdlA and rdlB genes, approximate transcription start sites, as determined by S1 nuclease mapping (Fig. 3), are shown in green, and putative −10 and −35 sequences are shown in red. The transcription start site for rsbN was assigned by S1 nuclease mapping (Fig. 5B) and in vitro transcription (Fig. S5). The transcription start site for the bldMp1 promoter was assigned by in vitro transcription (see Fig. 8A) and by alignment with the S. coelicolor bldMp1 transcription start site, which has been determined by S1 nuclease mapping (Bibb et al., 2000). The putative rdlC promoter sequence shown was identified by visual inspection and the 5′ end of the rdlC transcript has not been determined experimentally. B. Sequence logo for σBldN-dependent promoters, derived using WebLogo (http://weblogo.berkeley.edu). The height of the letters in the sequence logo, in bits, is proportional to the frequency of the A, C, T or G nucleotides at each position of the motif. This figure is available in colour online at http://wileyonlinelibrary.com.

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chpH and chpC lie adjacent to each other (Fig. 2), suggesting they might form an operon. For this reason, the radioactive probe used to analyse the intergenic region between chpH and chpC carried a 3′ non-homologous tail, which allows readthrough transcription to be distinguished from probe–probe re-annealing artefacts (Fig. 3). The upstream end of this probe lies 22 nucleotides inside the chpH-coding sequence, and the results therefore show that chpC is expressed by readthrough transcription, most likely from the chpH promoter. The data show that this readthrough transcription depends on bldN (Fig. 3), as expected from the microarray transcriptional profiling experiments (Fig. 1) and from the S1 nuclease protection analysis of the chpH promoter (Fig. 3). However, it should be noted that the possibility that the readthrough transcription arises from an uncharacterized promoter internal to the chpH-coding sequence cannot formally be excluded.

σBldN directs transcription of rsbN (sven3186), the gene lying immediately downstream of bldN

In addition to the chp and rdl genes, a further σBldN target gene identified in the ChIP-chip analysis was sven3186, here named rsbN (regulator of sigma-BldN), encoding a 43 kDa protein (Fig. 5A). rsbN lies immediately downstream of bldN, separated by an intergenic region of 298 nucleotides. Importantly, like the bldN gene itself (Elliot et al., 2001; den Hengst et al., 2010), rsbN was also shown by ChIP-chip to be a direct target of the transition state regulator BldD during vegetative growth in S. coelicolor (Fig. S3). To confirm and extend the σBldN ChIP-chip analysis, the 5′ end of the rsbN transcript was determined using S1 nuclease protection analysis. These experiments identified a single transcription start site lying 122 bp upstream of the rsbN GTG start codon in wild-type S. venezuelae, a transcript that was absent from the bldN mutant (Fig. 5B). The putative −10 and −35 sequences of this promoter again matched the σBldN consensus sequence logo established by the alignment of the chp, rdl and bldMp1 promoters (Fig. 4). As in the chpC S1 nuclease protection experiment, the mapping of the rsbN promoter was performed using a probe having a 3′ non-homologous tail to allow readthrough transcription to be distinguished from probe-probe re-annealing artefacts. In addition to identifying the rsbN promoter, this experiment clearly showed there was readthrough transcription from upstream (Fig. 5B). The upstream end of the probe used in Fig. 5B lies 30 bp downstream of the bldN stop codon, but a further experiment giving the same answer was performed using a probe with an upstream end 63 nucleotides inside the bldN-coding sequence (Fig. S4). These experiments show that rsbN is expressed from a second, upstream promoter, in addition to its own, dedicated promoter. It therefore seems likely that this readthrough transcription arises from the bldN promoter, which is not σBldN-dependent. The bldN promoter has been mapped in S. coelicolor (Bibb et al., 2000) and is well conserved at the sequence level in S. venezuelae. Thus, rsbN is transcribed by the EσBldN holoenzyme from its own dedicated promoter but also appears to be co-transcribed with bldN from the bldN promoter, even though the bldN and rsbN genes are separated by an intergenic region of nearly 300 nucleotides.

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Figure 5. A. σBldN ChIP-chip data for the rsbN promoter. The data for the bldN+ strain (blue dots) show averaged values from three independent biological replicates, whereas the data for the bldN null mutant control strain (green dots) are from a single experiment. DNA obtained from immunoprecipitation of σBldN was labelled with Cy3 and hybridized to DNA microarrays together with a total DNA control that was labelled with Cy5. The data are plotted as Cy3/Cy5 ratios (y-axis), as a function of chromosome location (x-axis). B. S1 nuclease mapping of the rsbN promoter and readthrough transcription from bldN. The size standards were a heat-denatured, [γ-32P]-labelled HinfI digest of ΦX174 DNA. This figure is available in colour online at http://wileyonlinelibrary.com.

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RsbN is a σbldN-specific anti-sigma factor

rsbN is a direct biochemical target of both σBldN and BldD and is in part co-transcribed with bldN. These observations suggested that RsbN is likely to play an important role in Streptomyces development, and we therefore disrupted rsbN to see if it had any obvious phenotypic consequences. Strikingly, deletion of rsbN accelerated the sporulation process in S. venezuelae. After 24 h on MYM solid medium, the wild type had erected aerial hyphae but had not yet started to sporulate, as revealed by the white appearance of the strain (Fig. 6) and by phase-contrast microscopy. In contrast, at 24 h, colonies of the rsbN null mutant had already synthesized the green polyketide pigment characteristic of S. venezuelae spores (Fig. 6) and microscopic examination confirmed that it had already sporulated extensively.

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Figure 6. Disruption of rsbN causes precocious sporulation. After 24 h on MYM solid medium, wild-type S. venezuelae is white, indicating that it has erected aerial hyphae but not yet started to sporulate. In contrast, at 24 h, colonies of the rsbN null mutant have already synthesized the green polyketide pigment characteristic of S. venezuelae spores, and microscopic examination confirmed that it had already sporulated extensively. This figure is available in colour online at http://wileyonlinelibrary.com.

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ECF sigma factors are very often controlled post-translationally by cognate anti-sigma factors and the genes encoding the ECF sigma factor and the anti-sigma factor typically form an operon with the anti-sigma factor gene lying downstream (Paget and Buttner, 2003; Campbell et al., 2007; 2008; Starońet al., 2009; Helmann, 2011). Given that σBldN is an ECF sigma factor and the phenotype of the rsbN null mutant was potentially consistent with RsbN serving to antagonize σBldN function, we carried out a series of experiments to determine if RsbN is a σBldN-specific anti-sigma factor.

First, we tested σBldN and RsbN for direct interaction in Escherichia coli using a bacterial two-hybrid (BACTH) system (Karimova et al., 1998). bldN was fused to the gene encoding the T18 fragment of adenylate cyclase in the vector pUT18 such that σBldN was at the N-terminus of the fusion protein, and was also fused to the gene encoding the T25 fragment of adenylate cyclase in the vector pKT25 such that σBldN was at the C-terminus of the fusion protein. Parallel pUT18 and pKT25 constructs were made carrying fusions to RsbN. Pairs of vectors carrying just one of the two genes were used as negative controls.

Interacting pairs of proteins were screened initially by transforming E. coli BTH101 with the appropriate plasmids and monitoring restoration of adenylate cyclase activity on MacConkey-maltose indicator plates. Interaction between BldN and RsbN was readily observed, regardless of which protein was fused to the T18 fragment of adenylate cyclase and which was fused to the T25 fragment, and therefore whether the fusions were N- or C-terminal. Clones of each pair were assayed for β-galactosidase activity, showing both arrangements yielded approximately equivalent enzymatic activity (∼ 800–900 units) (Fig. 7A). Both RsbN and σBldN failed to interact with themselves (Fig. 7A).

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Figure 7. A. Bacterial two-hybrid experiment to investigate the interaction between σBldN and RsbN. The listed pairs of constructs were transferred into the BACTH reporter strain E. coli BTH101 by transformation. The resulting transformants were selected on MacConkey-maltose agar containing 100 µg ml−1 carbenicillin and 50 µg ml−1 kanamycin and incubated at 30°C for several days before two independent clones were picked and subjected to β-galactosidase assays. The histogram was plotted using the average activity levels in Miller units of the two clones. B. Co-elution assay to investigate the interaction between σBldN and RsbN. Buffer A (0.5 ml) containing BSA (6 µm), His6-RsbN (120 nm) and non-tagged σBldN (240 nm) was incubated at 20°C for 10 min before being loaded onto a HIS-Select spin column. As a control, an equivalent 0.5 ml of σBldN-BSA sample that lacked RsbN was incubated in the same way and passed down a second HIS-Select nickel column. After extensive washing, retained proteins were eluted with imidazole and the flow-through (FT) and eluate (E) from each column were analysed by (i) SDS-polyacrylamide gel electrophoresis and (ii) immunoblotting. A sample of purified σBldN was added to the size standards (MW) to aid identification.

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We next attempted to overexpress and purify RsbN in E. coli to permit in vitro biochemical experiments. The complete rsbN gene was cloned into pET28a and RsbN was overexpressed in E. coli with an N-terminal hexa-histidine tag. Some membrane prediction programmes (e.g. TMPRED, Phobius) predict that RsbN would contain a single transmembrane helix, corresponding to residues 118–139 (see Discussion). Despite this, RsbN was found in the soluble fraction of the cell extract and was straightforwardly purified to homogeneity by nickel affinity chromatography followed by anion exchange. In contrast, like S. coelicolorσBldN (Bibb et al., 2000), S. venezuelaeσBldN formed inclusion bodies when overexpressed in E. coli, and had to be solubilized and refolded for the work described below, as described in Experimental procedures.

To see if RsbN and σBldN would interact in vitro, bovine serum albumin (BSA), His6-RsbN and non-tagged σBldN that had been solubilized from inclusion bodies and refolded, were mixed together in a 50:1:2 molar ratio and passed down a HIS-Select nickel column. After extensive washing, RsbN was eluted from the column with imidazole and non-tagged BldN was found to co-elute with it (Fig. 7B). As a control, an equivalent σBldN-BSA sample that lacked RsbN was passed down a second HIS-Select nickel column. σBldN passed straight through this column and none eluted with imidazole (Fig. 7B). Taken together, these experiments suggested that RsbN and σBldN form a complex.

To confirm and extend this initial experiment, we examined the effect of RsbN on σBldN-directed transcription in reactions containing purified S. venezuelaeσBldN, an S. venezuelae bldMp1 promoter template and E. coli core RNA polymerase. In a control experiment, we replaced σBldN with σR, the ECF sigma factor that mediates the disulphide stress response in Streptomyces (Paget et al., 1998; Paget and Buttner, 2003; den Hengst and Buttner, 2008; Park and Roe, 2008; Kim et al., 2009), and substituted the bldMp1 promoter with a DNA template containing the σR-dependent promoter sigRp2. RsbN inhibited σBldN-directed transcription, but equivalent amounts of RsbN had no effect on σR-directed transcription (Fig. 8A), showing that RsbN functions as a σBldN-specific anti-sigma factor.

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Figure 8. A. Inhibition of σBldN-directed in vitro transcription by RsbN. Purified RsbN (300, 100 or 10 nM final concentration) was added to the transcription buffer containing σBldN (100 nM), or σR (100 nM), and DNA templates (7.5 nM) containing bldMp1 (for σBldN) or sigRp2 (for σR). Transcripts were synthesized and analysed as described in Experimental procedures. B. Coexpression and co-purification of His6-RsbN and non-tagged σBldN. σBldN and N-terminally his-tagged RsbN were coexpressed in E. coli using the DUET expression vector system and the soluble extract was passed in tandem down a HiTrap FF 5 ml column and a HiLoad Superdex 75 26/60 gel filtration column. Final peak fractions were analysed by SDS-polyacrylamide gel electrophoresis and the identities of the two proteins were confirmed by tryptic mass fingerprinting.

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RsbN and σBldN form a stable complex in vivo

Finally, we coexpressed σBldN and N-terminally His-tagged RsbN using the E. coli DUET expression vector system. When S. venezuelaeσBldN was expressed by itself it formed 100% inclusion bodies. In contrast, when RsbN and σBldN were expressed together, all the σBldN was found in the soluble fraction of the extract. This is reminiscent of data from other systems in which proteins that are known to form part of a complex are insoluble when expressed in isolation but soluble when expressed with their cognate partner protein (e.g. the α and β subunits of lambda integrase; Nash et al., 1987). Further, when the soluble extract was passed in tandem down a nickel column and then a gel filtration column, σBldN co-purified with RsbN in approximately stoichiometric amounts, despite the fact that only RsbN was His-tagged (Fig. 8B). The stoichiometry of the two proteins was approximately constant across the peak, implying a stable complex. The identities of the two proteins were confirmed by tryptic mass fingerprinting. Thus, σBldN and its cognate anti-sigma factor RsbN can form a stable complex in vivo and in vitro.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

S. venezuelae as a new model species for developmental studies

The work described here is the first demonstration of the utility of S. venezuelae as a developmental system. S. venezuelae sporulates in liquid culture and > 90% total biomass is converted into spores. The S. venezuelae genome sequence was released in 2011, and the Affymetrix whole-genome chips used here and by Pullan et al. (2011) for microarray transcriptional profiling are available. Further, an ordered cosmid library covering the genome has been constructed (M. Bibb and M. Buttner, unpublished), permitting the ready application of ‘Redirect’ PCR-targeted gene disruption (Gust et al., 2003; 2004) to this species. Other potential benefits of this species as an experimental system include rapid, highly dispersed growth to high density, facilitating physiological work (Pullan et al., 2011), a robust generalized transduction system based on the phage SV1 (Stuttard, 1979), and the ability to introduce DNA by electroporation, allowing the application of ‘EZ-Tn5’ transposon mutagenesis (Epicentre Biotechnologies).

Expression of the chaplins and the rodlins

The chaplin and rodlin proteins were discovered and characterized in the context of aerial mycelium formation on agar plates. These discoveries were made using S. coelicolor, a species that does not sporulate in liquid culture. It is therefore informative to find that expression of the chp and rdl genes is activated as part of the sporulation cascade in S. venezuelae during differentiation in liquid culture. Submerged sporulation does not involve the growth of an aerial mycelium, so there is no requirement for hyphae to escape the aqueous milieu and grow into the air. Nevertheless, transcription of the chp and rdl genes is activated, implying that expression of the hydrophobic sheath is an obligatory part of the sporulation programme, regardless of whether it occurs on plates or in liquid culture.

Streptomyces griseus has an intermediate phenotype between S. coelicolor and S. venezuelae in that it sporulates weakly in liquid culture. Interestingly, the spores that are produced by liquid cultures of S. griseus are decorated with the rodlet ultrastructure characteristic of the hydrophobic sheath (Claessen et al., 2004). This suggests that, like S. venezuelae, S. griseus expresses its chp and rdl genes during submerged sporulation, a suggestion confirmed for one of these genes, rdlA (Claessen et al., 2004).

One possible explanation for hard-wiring expression of the chaplins and rodlins into the sporulation programme is that the hydrophobic sheath might be essential for the long-term viability of spores. In addition, the hydrophobic sheath may also be important for the distribution of spores. In the soil, it is thought that Streptomyces spores are dispersed, at least in part, on the surface of water droplets because they adhere to surface tension, a process that is mediated by the hydrophobic sheath (Elliot et al., 2008). To address these possibilities experimentally, it will be necessary to compare the viability, resistance and other characteristics of spores that do and do not have a hydrophobic sheath. It has not been possible to generate chaplin-less spores in S. coelicolor because strains deleted for all the chaplin genes cannot produce aerial hyphae on minimal media and are severely deficient on rich media, and therefore do not make spores (Claessen et al., 2003; Capstick et al., 2007). In contrast, it should be feasible to address these issues in S. venezuelae because it can sporulate in liquid culture without the need for aerial mycelium formation. As a consequence, it should be possible to generate a strain of S. venezuelae deleted for all the chaplin genes and, through submerged sporulation, use it to generate chaplin-less spores. Alternatively, if it turns out that a chaplin-less mutant of S. venezuelae cannot sporulate in liquid, it would imply that the assembly of the hydrophobic sheath acts as a morphological checkpoint that couples the formation of the amyloid chaplin filaments on the surface of the developing spores to the regulatory cascade.

The functions of the chaplins and rodlins partially overlap with those of the surfactant peptide SapB, which is specified by the ram genes. Transcription of the ram genes is not significantly upregulated during submerged sporulation in wild-type S. venezuelae under the conditions used here (data not shown), suggesting that expression of SapB is not an obligatory part of the sporulation programme of S. venezuelae in the same way as expression of the chp and rdl genes. This is consistent with studies in S. coelicolor, which showed that SapB expression is conditionally associated with differentiation, occurring on rich media but not on minimal media (Willey et al., 1991). Further, the ChIP-chip experiments reported here show that the ram genes are not direct regulatory targets of σBldN.

Transcriptional organization of the bldN–rsbN operon

bldN and rsbN are conserved and adjacent in each of the 28 publicly available streptomycete genome sequences we have examined, with the intergenic gap varying in size between 200 bp (in Streptomyces griseoflavus Tü4000) and 320 bp (in Streptomyces roseosporus NRRL 15998). The transcriptional organization of the bldN–rsbN operon and that of a canonical ecf-anti-sigma pair involved in a classic stress response are shown diagrammatically in Fig. 9 (Paget and Buttner, 2003; Starońet al., 2009; Helmann, 2011). Typically, the start and stop codons of the genes encoding an ECF sigma factor and its cognate anti-sigma factor overlap in the ‘ATGA’ manner, presumably to ensure the two proteins are produced in a 1:1 stoichiometry. Also, expression of both genes is typically driven from two promoters lying upstream of the operon, one a relatively weak constitutive promoter and the other a promoter under the control of the sigma factor itself. This autoregulatory promoter leads to a rapid spiral of auto-induction when the signal inactivating the anti-sigma is received, ensuring a dramatic response to the perceived stress (Helmann, 1999; 2002; 2011; Paget and Buttner, 2003; Starońet al., 2009). In contrast, the rsbN gene is separated from bldN by a 298 bp intergenic region, and the σBldN target promoter lies in this gap, not upstream of both genes, meaning that σBldN directs transcription of its cognate anti-sigma factor but does not activate its own expression. Perhaps this reflects the fact that σBldN is involved in the control of sporulation (Bibb et al., 2000), and not a classic stress response such as the one mediated by σR in response to oxidative stress, where a dramatic and immediate response is required to inactivate oxidizing agents and repair cellular damage in order to minimize cell death (Paget et al., 1998; Paget and Buttner, 2003; den Hengst and Buttner, 2008; Park and Roe, 2008; Kim et al., 2009).

image

Figure 9. Diagrammatic representation of the transcription organization of (A) the bldN–rsbN operon and (B) a typical ecf-anti-sigma operon.

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The bldN promoter, which drives expression of both bldN and rsbN, is upregulated during development and this de-repression arises, at least in part, because the promoter is repressed by the transition state regulator BldD during vegetative growth, at least in S. coelicolor (Elliot et al., 2001; den Hengst et al., 2010). Similarly, the rsbN promoter is also a target of BldD during vegetative growth (Fig. S3) (den Hengst et al., 2010). Whether the bldN promoter is also subject to activation in S. venezuelae or S. coelicolor is unknown. However, adsA, the orthologue of bldN in S. griseus, is directly activated by AdpA (BldH), and is therefore under the control of the well-characterized A-factor δ-butyrolactone signalling cascade that controls both differentiation and antibiotic production in that species (Yamazaki et al., 2000; Chater and Horinouchi, 2003; Ohnishi et al., 2005; Horinouchi, 2007; 2008; Flärdh and Buttner, 2009).

RsbN is a novel anti-sigma factor

There are probably two principal reasons why rsbN has not previously been investigated experimentally, despite lying next to bldN. First, as described above, rsbN is not translationally coupled to bldN, as is typical for most ecf-anti-sigma pairs, but is instead well separated from bldN. The second reason is that blast searches with RsbN do not identify any known anti-sigma factors. It is common for bacteria to express alternative σ factors before they are needed, but to maintain them in an inactive state using anti-σ factors until their release is triggered by appropriate conditions. However, whereas all members of the σ70 family are homologous, anti-σ factors belong to a number of phylogenetically unrelated families. In the comprehensive bioinformatic analysis of ECF sigma factors conducted by Starońet al. (2009), σBldN was assigned to the minor group ECF121, containing only eight members at that time, five of which are encoded adjacent to genes encoding uncharacterized proteins showing some degree of similarity to RsbN. Of these five sigma factors, only σD from Mycobacterium tuberculosis has been investigated experimentally (Raman et al., 2004), but this did not include any investigation of Rv3414c, the protein showing weak similarity to RsbN.

Membrane prediction programmes such as Phobius (http://phobius.sbc.su.se/) and TMPRED (http://www.ch.embnet.org/software/TMPRED_form.html) predict that RsbN would contain a single transmembrane helix, corresponding to residues 118–139 (GLAAGGLTVGVAAGAFGGVAAA), with the N-terminal 117 residues forming a 12.5 kDa cytoplasmic domain and the bulk of the protein, residues 140–412, forming a 29 kDa extracytoplasmic domain. Despite this, when overexpressed in E. coli, RsbN was found in the soluble fraction of the cell extract. Further, when RsbN was co-overexpressed with σBldN, RsbN prevented σBldN from forming inclusion bodies and the two proteins formed a stable, soluble complex, showing that the overexpressed RsbN was active as an anti-sigma factor. These results raise the possibility that the analysis of RsbN using the membrane prediction programmes is misleading. However, we have not yet determined whether RsbN is soluble or membrane-associated when expressed at native levels in S. venezuelae.

What is now clear from the work described here is that σBldN plays a pivotal role in the developmental cascade, directly activating expression of the chp and rdl genes, in addition to bldM. Therefore, regardless of whether RsbN is cytoplasmic or a transmembrane protein, a major future challenge in Streptomyces developmental biology will be to determine how the activity of the RsbN anti-sigma factor is regulated, and to identify the signals that lead to its inactivation during differentiation.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains, growth conditions and conjugations

Escherichia coli K-12 strain DH5α was used for plasmid and cosmid propagation. BW25113 (Datsenko and Wanner, 2000) containing a λ RED plasmid, pIJ790, was used to create the disruption cosmid. Cosmids were conjugated from the dam dcm hsdS E. coli strain ET12567 containing pUZ8002 (Paget et al., 1999) as described by Gust et al. (2003; 2004). Although ET12567 was used for this purpose, it should be noted that S. venezuelae does not restrict methylated DNA, and so cosmids can be conjugated from methylating E. coli strains. S. venezuelae strains used are summarized in Table 1 and were cultured in MYM (Stuttard, 1982) made with 50% tap water and supplemented with 200 µl of trace element solution (Kieser et al., 2000) per 100 ml.

Table 1. Strains and plasmids used in this study.
Strains/plasmidsRelevant genotype/commentsSource or reference
Strains
S. venezuelae   
 ATCC10712The wild type 
 SV12ΔbldN::aprThis study
 SV15ΔrsbN::aprThis study
E. coli   
 ET12567 (pUZ8002)ET12567 containing helper plasmid pUZ8002 Paget et al. (1999)
 BW25113Δ(araD-araB)567ΔlacZ4787(::rrnB-4) lacIp-4000(lacIQ), λ-rpoS369(Am) rph-1Δ(rhaD-rhaB)568 hsdR514 Datsenko and Wanner (2000)
Plasmids
 pIJ773Plasmid template for amplification of the apr oriT cassette for ‘Redirect’ PCR-targeting Gust et al. (2003)
 pIJ790Modified λ RED recombination plasmid [oriR101][repA101(ts)]araBp-gam-be-exo Gust et al. (2003)
 pMS82Plasmid cloning vector for the conjugal transfer of DNA from E. coli to Streptomyces spp. Integrates site specifically at the ΦBT1 attachment site (HygR) Gregory et al. (2003)
 pIJ6767pMS82 carrying bldN driven from its own promoterThis study
 pIJ6768pMS82 carrying rsbN driven from its own promoterThis study
 pIJ6769 bldN cloned in the bacterial two-hybrid vector pUT18CThis study
 pIJ6770 bldN cloned in the bacterial two-hybrid vector pKT25This study
 pIJ6771 rsbN cloned in the bacterial two-hybrid vector pUT18CThis study
 pIJ6772 rsbN cloned in the bacterial two-hybrid vector pKT25This study
 pIJ6773 bldN cloned in pUC57This study
 pIJ6774 bldN cloned in the expression vector pET20bThis study
 pIJ6775 rsbN cloned in the expression vector pET28bThis study
 pIJ6776 bldN cloned in the expression vector pETDuet-1This study
 pIJ6777 rsbN and bldN cloned in the expression vector pETDuet-1This study

Conjugations between E. coli and S. venezuelae were carried out as described for S. coelicolor (Kieser et al., 2000) but with two differences. First, because S. venezuelae grows much more quickly than S. coelicolor, the plates were incubated either at room temperature overnight or at 30°C for only 7 h before overlaying with the selective antibiotics (incubation at 30°C for longer periods before overlaying results in confluent lawns of S. venezuelae). Second, transconjugants were selected on R2-S medium [R2 medium (Kieser et al., 2000) lacking both sucrose and glucose but containing 1% (w/v) maltose].

Construction and complementation of S. venezuelae null mutants

Using the ‘Redirect’ PCR targeting method of Gust et al. (2003; 2004), bldN and rsbN and mutants were generated in which the coding region was replaced with a single apramycin-resistance (apr) cassette. A cosmid library that covers > 95% of the S. venezuelae genome (M. Bibb et al., unpublished) is fully documented at http://strepdb.streptomyces.org.uk/. Cosmid Sv-5-D01 was introduced into E. coli BW25113 containing pIJ790 and the relevant genes were replaced with the apr-oriT cassette amplified from pIJ773 using the primer pairs bldNdisfor and bldNdisrev, or rsbNdisfor and rsbNdisrev (Table S1). The resulting disrupted cosmids were confirmed by restriction digestion and by PCR analysis using flanking primers, and introduced into S. venezuelae by conjugation. Null mutant derivatives, generated by double crossing over, were identified by their apramycin-resistant, kanamycin-sensitive and morphological phenotypes, and their chromosomal structures were confirmed by PCR analysis using flanking primers and by Southern hybridization using the entire cosmid Sv-5-D01, partially digested with Sau3A1, as a probe. Representative null mutants were designated SV12 (bldN) and SV15 (rsbN). The growth rates of the mutants in MYM were comparable to that of the wild type. For complementation, bldN was amplified with the primers bldNcompfor and bldNcomprev, generating a fragment carrying the coding sequence and the bldN promoter, and cloned into HindIII-cut pMS82 (Gregory et al., 2003) to create pIJ6767. rsbN was amplified with the primers rsbNcompfor and rsbNcomprev, generating a DNA fragment carrying the coding sequence and the rsbN promoter, and cloned into EcoRV-cut pMS82 (Gregory et al., 2003) to create pIJ6768. The plasmids were introduced into the respective mutants by conjugation and, in each case, the mutant phenotype was fully complemented in trans.

RNA isolation and DNA microarray analysis

For RNA isolation, triplicate MYM cultures (supplemented with trace element solution) were grown with shaking (250 r.p.m.) at 30°C and samples were collected at 2 h intervals from 8 to 20 h (26 ml at 8 h, 9 ml at 10 h and 6 ml at subsequent time points). A 300 µl sample was removed at each time point to check the stage of development by phase-contrast light microscopy, and to measure the optical density. Cells were harvested by centrifugation at 4°C, frozen in dry ice/ethanol, and ground in liquid nitrogen in a mortar and pestle that had been chilled in a dry ice/liquid nitrogen bath. Ground material was suspended in 2 ml of TRIzol (Invitrogen) and divided equally between two 2 ml tubes; one was stored at −80°C as a backup and the other used for RNA isolation. Total RNA was isolated using the RNeasy mini kit (Qiagen), essentially as described by Hesketh et al. (2009).

Single-strand reverse transcription and indirect labelling of 10 µg of total RNA were performed for hybridization to Streptomyces diS_div712a GeneChip arrays according to the manufacturer's published protocol (Affymetrix). GeneChips were washed and stained using a GeneChip fluidics workstation model 450, and scanned with a Gene Array Scanner. After pre-processing, the data were imported into GeneSpring 7.3 (Agilent Technologies), converted to log2 values and normalized per gene to the median. Error models based on replicate values were implemented and statistical calculations on the filtered data were performed in GeneSpring by two-way anova as described previously (Hesketh et al., 2009). Transcriptome data have been deposited at the MIAME-compliant ArrayExpress database under Accession No. E-MEXP-3612.

ChIP-chip analysis

Culture conditions for ChIP-chip analysis were the same as those for microarray analysis, and samples were taken for chromatin immunoprecipitation at time points between 12 and 14 h. Immunoprecipitations were performed as described by den Hengst et al. (2010) but using a σBldN-specific polyclonal antibody. Three replicates were performed for the rsbN mutant and the bldN mutant control experiment was performed once. Qualitatively similar results to those obtained with the rsbN mutant were observed using wild-type S. venezuelae, but the level of DNA binding was enhanced in the rsbN mutant, presumably because no σBldN is sequestered by RsbN in this background.

DNA labelling and hybridization to DNA microarrays were performed by Oxford Gene Technology (OGT) and were carried out essentially as described previously (den Hengst et al., 2010; Pullan et al., 2011). Briefly, using the Bio-Prime kit (Invitrogen), 800 ng of the total and immunoprecipitated DNA was labelled with Cy5-dCTP and Cy3-dCTP respectively. Labelled DNA was hybridized, in an Agilent Technologies hybridization oven, to high-density DNA microarrays representing the genome of S. venezuelae (GenBank Accession No. FR845719), which were designed and manufactured by OGT. Following washing, the arrays were read using an Agilent Technologies scanner and the Cy5 and Cy3 signals were quantified using Agilent's Feature Extraction software. From the data files received from OGT, the green median signal intensities and the red median signal intensities were read into a data frame in R, converted to log (base 2) and green to red signal log ratios calculated. These log(2) ratios were normalized to the average of the medians of red and green signal intensities using the loess function of R. If there were replicates, the log(2) ratios for each probe were averaged to a single mean. The (means of) log(2) ratios were smoothed using the smooth function of R with default settings and converted to simple ratios before plotting the final figures. ChIP-chip data have been deposited at the MIAME-compliant ArrayExpress database under Accession No. E-MTAB-1103.

Identification of DNA motifs

To identify conserved DNA motifs, the DNA sequences of the promoter regions of the bldM, chp, rdl and rsbN genes were collated from the genome sequence of S. venezuelae (GenBank Accession No. FR845719) and this data set was used as input for the MEME software tool (Bailey and Elkan, 1994) to search for motifs.

Two-hybrid analysis

Two-hybrid analyses were performed essentially as described by Foulston and Bibb (2011). pKT25 contains a multiple-cloning site (MCS) preceded by a fragment of the Bordetella pertussis cya gene that encodes half of the catalytic domain (T25) of adenylate cyclase (CyaA) (Karimova et al., 1998). This allows generation of N-terminal fusion proteins. The other half of CyaA (T18) is encoded by pUT18 and the MCS precedes the cya gene fragment, allowing the generation of C-terminal fusion proteins (Karimova et al., 2001). bldN was PCR-amplified with primers bldN_18for and bldN_18rev (no stop codon included; Table S1), cloned into SmaI-cut pUC19, excised with XbaI and EcoRI and cloned into XbaI–EcoRI-cut pUT18 to generate pIJ6769. bldN was amplified a second time with primers bldN_25for and bldN_25rev (stop codon included; Table S1), cloned into SmaI-cut pUC19, excised as an XbaIEco53KI fragment and cloned into XbaI–SmaI-cut pKT25 to create pIJ6770. rsbN was amplified with primers rsbN_18for and rsbN_18rev, gel purified, digested with EcoRI and HindIII, and cloned into EcoRI–HindIII-cut pUT18 to generate pIJ6771. rsbN was also amplified with primers rsbN_25for and rsbN_25rev (Table S1), digested with BamHI and Asp718 and cloned into BamHI–Asp718-cut pKT25 to generate pIJ6772. All constructs were confirmed by sequencing.

Combinations of the resulting plasmids were introduced into E. coli BTH101 by co-transformation and screened on MacConkey medium (Difco) containing 1% maltose, 0.5 mM IPTG (isopropyl-beta-d-thiogalactopyranoside), 50 µg ml−1 kanamycin and 100 µg ml−1 carbenicillin. Pairs of vectors carrying just one of the two genes were used as negative controls. Plates were incubated at 30°C for several days until the colour of the colonies had developed fully. Quantification of β-galactosidase activities was carried out as previously described (Slavny et al., 2010). Two independent clones from each interaction plate were assayed and the values were averaged.

S1 nuclease protection analysis

RNA was purified from wild-type S. venezuelae as described above. Probes for S1 nuclease protection analysis were generated by PCR using the oligonucleotide pairs shown in Table S1. Non-homologous tails (underlined) were incorporated into some probes to distinguish between full-length protection and probe/probe re-annealing artefacts. The downstream (reverse) primer in each case was 5′ end-labelled using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]-ATP (PerkinElmer), as described by the manufacturer. S1 nuclease mapping was carried out as described previously (Bibb et al., 2000). The size standards were a heat-denatured, [γ-32P]-labelled HinfI digest of ΦX174 DNA (Promega).

Overexpression and purification of σBldN, RsbN and the σBldN–RsbN complex

A truncated version of the S. venezuelae bldN gene starting at Met-84 (and therefore lacking the unusual σBldN N-terminal extension) was chemically synthesized with optimal E. coli codon usage, flanked by a 5′ NdeI site and a 3′ XhoI site (Genescript), and cloned into pUC57 to create pIJ6773. bldN was excised from pIJ6773 as an NdeIXhoI fragment and cloned into NdeIXhoI-cut pET20b (Novagen) to generate pIJ6774, or into NdeIXhoI-cut pETDuet-1 (Novagen) to generate pIJ6776. rsbN was PCR-amplified using primers rsbN_overexpfor and rsbN_overexprev (Table S1), cloned into SmaI-cut pUC19, sequenced, and then excised using NdeI and HindIII sites introduced during PCR. This fragment was cloned into pET28b to generate pIJ6775. rsbN was excised from pIJ6775 as an XbaIHindIII fragment and cloned into pIJ6776 to generate the σBldN–RsbN coexpression construct, pIJ6777.

pIJ6774 and pIJ6775 were introduced into BL21(DE3)pLysS for protein expression. S. venezuelaeσBldN was overexpressed and purified from inclusion bodies as described previously for S. coelicolorσBldN (Bibb et al., 2000). BL21(DE3)pLysS carrying pIJ6775 was grown at 37°C in L broth to an OD600 of 0.5 when the cells were briefly chilled on ice, induced with 0.5 mM IPTG and then grown at 30°C for a further 3 h. The cells were spun and washed, resuspended in Buffer A [50 mM Tris, pH 8.0, 0.5 M NaCl, 30 mM imadazole, 5% (v/v) glycerol] and subjected to sonication for 30 s at half power over 7 cycles, with 1 min on ice in between. The cell debris was then removed by centrifugation (15 000 r.p.m., 4°C, 15 min) and soluble N-terminally His-tagged RsbN was purified from the supernatant. The supernatant was filtered through a 0.45 micron filter, loaded on a HiTrap HP column (GE Healthcare) using an ÄKTA HPLC P900 (Amersham Biosciences) and eluted using a 30 mM to 500 mM imadazole gradient in Buffer A. Fractions containing RsbN were pooled, diluted 50-fold with Buffer C (20 mM Tris, pH 7.5,10 mM NaCl), loaded onto an Anion Q FF 5 ml column (GE Healthcare) and eluted using a 10 mM to 1 M NaCl gradient in Buffer C. The σBldN–RsbN complex was purified from BL21(DE3)pLysS containing pIJ6777 using an ÄKTAxpress™ HPLC system (GE Healthcare). Protein was eluted from a HiTrap FF 5 ml column (GE Healthcare) using Buffer B (Buffer A with 500 mM imadazole), and run directly into a HiLoad Superdex 75 26/60 gel filtration column that was eluted with GF Buffer (50 mM HEPES, pH 7.5, 100 mM NaCl).

Co-elution assay

Buffer A (0.5 ml) containing BSA (6 µm), His6-RsbN (120 nm) and non-tagged σBldN (240 nm) was incubated at 20°C for 10 min before being loaded onto a HIS-Select spin column (Sigma H7787) as per the manufacturer's instructions. After extensive washing with Buffer A, retained proteins were eluted using Buffer B (Buffer A with 500 mM imidazole). As a control, an equivalent 0.5 ml of σBldN-BSA sample that lacked RsbN was incubated in the same way and passed down a second HIS-Select nickel column.

In vitro transcription

In vitro runoff transcription assays were performed using [α-32P]-CTP (PerkinElmer) at 3000 Ci mmol−1 as described by Buttner et al., (1987). The S. venezuelae bldMp1 promoter template was PCR-generated with primers bldM_runofffor and bldM _runoffrev, S. venezuelae rsbN promoter template 1 with primers rsbN_S1for and rsbN_S1newrev, S. venezuelae rsbN promoter template 2 with primers rsbN_S1for and rsbN_S1rev, and the S. coelicolor sigRp2 promoter template with primers sigRp2_runofffor and sigRp2_runoffrev (Table S1). Transcripts were analysed on 6% polyacrylamide–7 M urea gels using a heat-denatured, [γ-32P]-labelled HinfI digest of ΦX174 as the size standards. E. coli core RNA polymerase was purchased from Epicentre Technologies (Madison, WI), and S. coelicolorσR was the kind gift of Mark Paget.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Anna Staroń, Thorsten Mascher and Tracy Palmer for helpful discussion, Clare Stevenson for instruction in using the AKTA Express, Lucy Foulston for help with β-galactosidase assays, Mark Paget for the gift of S. coelicolorσR, and OGT for expert handling of the ChIP samples. This work was funded by the MET Institute Strategic Programme Grant to the John Innes Centre from the BBSRC.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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