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. Author manuscript; available in PMC: 2009 Oct 7.
Published in final edited form as: Mol Microbiol. 2008 Aug;69(3):765–779. doi: 10.1111/j.1365-2958.2008.06330.x

Activation of Transcription Initiation by Spx: Formation of Transcription Complex and Identification of a Cis-acting Element Required for Transcriptional Activation

Dindo Y Reyes 1,†, Peter Zuber 1,*
PMCID: PMC2758557  NIHMSID: NIHMS65534  PMID: 18687074

Summary

The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP σA subunit and the −10 promoter sequence of trxA and B, and contact of the ββ′ subunits with core promoter DNA. No Spx-DNA contact was detected. Spx mutants, SpxC10A and SpxG52R, or RNAP α C-terminal domain mutants that impair productive Spx-RNAP interaction did not induce heightened σ and ββ′ contact with the core promoter. Deletion analysis and the activity of hybrid promoter constructs having upstream trxB DNA fused at positions −31, −36, and −41 of the srf (surfactin synthetase) promoter indicated that a cis-acting site between −50 and −36 was required for Spx activity. Mutations at −43 and −44 of trxB abolished Spx-dependent transcription and Spx-induced cross-linking between the σ subunit and the −10 region. These data are consistent with a model that Spx activation requires contact between the Spx/RNAP complex and upstream promoter DNA, which allows Spx-induced engagement of the σ and large subunits with the core promoter.

Keywords: Spx, Bacillus subtilis, trxB, trxA, transcription, cross-linking

Introduction

Positive control of transcription initiation is most often exerted by a nucleotide sequence-specific, DNA-binding protein that interacts with upstream promoter DNA to assist RNAP interaction with the core promoter elements. Transcriptional activation has been studied in exquisite detail in the cases of class I and class II positive control exerted by activators such as CRP-cAMP (Lawson et al., 2004). However, a number of regulators can stimulate transcription initiation without interacting with DNA in a sequence-specific manner. The DksA protein can exert positive and negative control on transcription in conjunction with the stringent factor (p)ppGpp (Kang and Craig, 1990; Paul et al., 2004; Paul et al., 2005), although in a recent study, the two factors were observed to act differentially to exert opposite effects on transcription (Åberg et al., 2008). Phage N4 gene expression requires a phage specific protein, N4SSB, that contacts the β′ subunit to stimulate transcription of the late phage genes in a nucleotide sequence-independent fashion (Miller et al., 1997). The AsiA protein of phage T4 alters the organization of the Escherichia coli host RNAP holoenzyme by contacting region 4 of the σ subunit, disrupting σ’s interaction with the flap domain of the β subunit, and thus making σ available for interaction with the phage-specific activator, MotA (Baxter et al., 2006; Hinton et al., 2005; Pal et al., 2003). Such RNAP-binding factors, recently given the term “appropriators”, alter holoenzyme architecture to induce changes in the global transcriptional cross-section (Campbell et al., 2008). SoxS is an activator with similar properties to the above regulatory proteins in that its primary target is RNAP, binding to which initiates the activation process according to the “pre-recruitment” model (Griffith and Wolf, 2004; Shah and Wolf, 2004). The SoxS-RNAP complex then engages the SoxS-controlled promoter via nucleotide-specific contacts between SoxS and the control region of the activated gene. Such factors control transcription initiation by altering promoter binding properties of RNAP, changing the stability of the open complex, or affecting some other step of initiation subsequent to close complex formation (Hinton et al., 2005; Miller et al., 1997; Rutherford et al., 2007).

The RNAP-binding protein Spx activates and represses transcription initiation in B. subtilis and very likely in other low-GC Gram positive bacteria (Zuber, 2004). Spx does not resemble structurally other transcription factors, but instead is a member of the ArsC (arsenate reductase) family of proteins (Martin et al., 2001; Zuber, 2004). Spx represses activator-stimulated gene transcription by interaction with the C-terminal domain of the RNAP αsubunit (αCTD), where binding prevents interaction between activator and RNAP αCTD (Nakano et al., 2003b; Zhang et al., 2006). Spx-RNAP interaction is also required for positive transcriptional control of genes whose products function in alleviating thiol-specific oxidative stress (Nakano et al., 2003a; Nakano et al., 2005). Notable among the genes activated by Spx are the thioredoxin and thioredoxin reductase genes (trxA and trxB, respectively), the products of which function ubiquitously as antioxidants in nature (Holmgren et al., 2005). In order to optimally activate transcription, Spx not only must interact with RNAP, but it also must be in an oxidized form in which its N-terminal CXXC motif is in the disulfide state (Nakano et al., 2005). Spx shows no sequence-specific DNA-binding activity [(Nakano et al., 2005) and see below], although it is possible that when bound to RNAP, a cryptic DNA-binding surface is unmasked and participates in the interactions between RNAP and promoter DNA.

In the study reported herein, the organization of the Spx-activated transcription complex was explored using nucleotide-specific DNA-protein cross-linking and by promoter mutational analysis to obtain a greater understanding of the mechanism by which Spx stimulates transcription initiation. The identification of a cis-acting element required for Spx-dependent transcriptional activation is also presented. The evidence presented below is consistent with a model that Spx-activated promoters possess upstream DNA to which the Spx/RNAP complex interacts, which promotes Spx-induced contact between the RNAP σ subunit, the ββ′ subunits and DNA of the core promoter elements.

Results

Spx influences the architecture of RNAP during transcription initiation of trxA and trxB

We have previously shown that the oxidized form of Spx is required for transcriptional activation of trxA and trxB (Nakano et al., 2005). While activation had been shown to involve disulfide formation in Spx, and its interaction with RNAP (Nakano et al., 2003a; Newberry et al., 2005), no DNA-binding activity associated with Spx protein had been detected [(Nakano et al., 2005), and see below]. To begin to elucidate the mechanism of Spx-dependent transcriptional activation of trxA and trxB, specific interactions between promoter DNA and the Spx/RNAP complex were explored by nucleotide-specific protein-DNA cross-linking analysis (Bartlett et al., 2000; Naryshkin et al., 2000; Renfrow et al., 2004). The nucleotide positions selected were located in regions that were protected from DNase I by Spx-RNAP interaction (Nakano et al., 2005). DNA probes were synthesized that bear azidophenacyl bromide (APB)-derivatized nucleotides at the selected positions in the promoter regions (Fig. 1A). The cross-linking probe is completed by incorporation of a 32P-labeled deoxynucleotide near the position bearing the cross-linker, followed by extension to synthesize the double-stranded, derivatized fragment. Modification of the targeted nucleotides by the UV-activated cross-linker was monitored and confirmed by gel electrophoresis (Fig. S1). Cross-linking reactions with RNAP purified from an spx deletion mutant were conducted in the presence of wild-type or mutant Spx derivatives that were shown previously to be inactive in transcriptional activation of trxA and trxB (Nakano et al., 2003a; Nakano et al., 2003b; Nakano et al., 2005). Reactions were incubated either on ice (0°C) or at 37°C, at which RNAP and promoter DNA would likely be in the closed or open complex, respectively. Following cross-linking and SDS gel electrophoresis, phosphorimaging revealed protein cross-linked to DNA as radiolabeled bands of protein, but also a band of unknown identity running at a position in the gel below that of the RNAP α subunit. It was noticed that this band appeared in reactions containing no added protein (Fig. S2), indicating that it was an artifact of the cross-linking procedure. The cross-linking data shows the cross-linked RNAP subunits that migrate to positions on the gel above the artifact band.

Figure 1.

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Effect of wild-type or mutant Spx on RNAP-promoter DNA cross-linking. (A) DNA sequence of the trxA and trxB promoters used in the cross-linking experiment. Highlighted in bold letters and numbered according to position relative to the transcription start site (+1) are the nucleotides that were modified by the UV-activated crosslinker, APB. Underlines and double underlines indicate regions protected by Spx/RNAP and the RNAP α subunit, respectively, from DNase I in previously reported footprinting experiments (Nakano et al., 2005). Nucleotides with a single grey underline are the start sites of transcription. Results of the cross-linking experiments for trxA (B), trxB (E) and rpsD (F) with His-tagged RNAP derived from spx deletion strain, as performed according to the Experimental Procedures, are shown. C and D show the quantification of ββ′ crosslinking at −1 and −7 of trxA, expressed as large subunit band intensity over α subunit band intensity. A final concentration of 300 nM for wild-type (WT), SpxC10A (C) or SpxG52R (G) mutant Spx were added to the reactions containing 75 nM RNAP, while reaction without Spx is indicated by 0. Conditions favoring open complex formation were created by incubation of reactions at 37°C for 30 min whereas closed complex was examined in reactions kept at on ice (0°C) for 30 minutes. Cross-linked products were resolved using 12% SDS-PAGE. For B, E and F, the numbers above each panel represents the cross-linking sites probed for each reaction. Positions on the gel corresponding to the bands of β, β′, α, and σ are indicated. (The artifact band mentioned in Supplementary Figure 2 is not shown in these gels figures of cross-linked protein and DNA).

In the absence of Spx, the RNAP α subunit and, to a lesser extent, the large subunits (ββ′) showed cross-linking at most of the derivatized residue positions. This is likely due to non-specific RNAP-DNA interaction as previously observed (Naryshkin et al., 2000). Addition of wild-type Spx to the reaction triggered significant interaction of the major sigma factor, σA with the −12 of trxA (Fig. 1B) at 0°C and 37°C. Spx-induced interaction of σA at −11 of trxB (Fig. 1E) was observed only at 37°C. One important difference between the two promoters is that trxA bears a TG-extended −10 region (Burr et al., 2000; Camacho and Salas, 1999; Helmann, 1995). These results suggest that wild-type Spx influences transcription initiation by facilitating efficient binding of σA to the −10 core promoter element. In reactions containing Spx, there was also enhanced contact of the ββ′ subunits with −1, −7, and −22 nucleotide positions in the trxA promoter (Fig. 1B), and with probes −4 and −21 of trxB (Fig. 1E). The enhanced cross-linking of the large RNAP subunits was only observed in reactions incubated at 37°C, in which RNAP is expected to be bound to DNA in the open complex. To quantify the Spx-induced ββ′ binding to promoter DNA, the band intensities of the large subunits cross-linked to trxA positions −1 and −7 were expressed as a ratio of ββ′ intensity to −1 and −7 cross-linked α subunit band intensity (Fig. 1C and D). In three independent reactions, enhanced ββ′ crosslinking was observed at 37°C when Spx was present.

Spx mutant proteins defective in redox-dependent activation (SpxC10A) (Nakano et al., 2005) or in contacting αCTD of RNAP (SpxG52R) (Nakano et al., 2003b; Newberry et al., 2005) were also tested for their effects on RNAP-promoter cross-linking. Reactions containing the mutant proteins showed reduced contact between promoter DNA and RNAP subunits σ and ββ′, compared to the cross-linking reactions containing wild type Spx (Fig 1B, C, D, and E). Notably, there was little evidence of σA contacting the −12 or −11 positions of trxA and trxB, respectively when the reactions contained the mutant Spx protein.

Cross-linking reactions containing a control promoter, rpsD [encoding ribosomal protein S4, (Grundy and Henkin, 1990)], which does not require a transcription factor for transcription initiation, as expected did not show any differences in cross-linked products when Spx was added to the reaction (Fig. 1F). An intense band of cross-linked σA protein to the −12 nucleotide position is detected in the gel of the reactions incubated at 37°C either in the presence or absence of Spx.

Residue substitutions in the αCTD domain impair Spx-dependent RNAP interaction with trxA and trxB promoter DNA

The rpoAcxs-1 mutation (rpoAY263C) results in a Y263C substitution in the helix 1 region of the αCTD (Nakano et al., 2000) that is part of the interaction interface of the Spx- αCTD complex (Newberry et al., 2005). The residue substitution disrupts Spx-αCTD interaction, thus impairing Spx-dependent activation of trxA and trxB transcription initiation. Likewise, the rpoAK267A mutant, generated through alanine-scanning mutagenesis, exhibited hypersensitivity to diamide-induced thiol-specific oxidative stress, and had a reduced level of Spx-activated trxB transcription (Zhang et al., 2006). As shown in Fig. 2A, contact was observed between the mutant RNAP holoenzymes and trxA promoter at each nucleotide position at 0°C, comparable to reactions containing wild-type RNAP except for the complete absence of mutant RNAP σA cross-linking at the trxA −10 promoter element (−12 in trxA). Incubation at 37°C resulted in loss of α subunit-DNA contact at several nucleotide positions as indicated by the reduced amount of cross-linked products in the presence or absence of Spx. The results indicate that the Y263 and K267 residues function in nucleotide-nonspecific RNAP-DNA interaction as well as in Spx-RNAP interaction. These functions of B. subtilis α subunit, both activator and DNA-binding, are similar to those of the binding surface of E. coli α that contacts the activator, SoxS (Shah and Wolf, 2004). In the case of RNAPK267A, contact between RNAP and promoter DNA at 0°C was observed, as was shown in reactions containing RNAPY263C. As with RNAPY263C, there was a reduction in contact between RNAPK267A and DNA at several positions (−15, −22, −30, −47, and −54) in reactions incubated at 37°C. The reduced nucleotide-nonspecific contact between mutant RNAP and DNA at the 37°C might account, in part, for the impaired ability of Spx to transcriptionally activate trxA. A role for nonspecific α and DNA contact in RNAP-promoter interaction has been reported (Ross and Gourse, 2005).

Figure 2.

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Effect of rpoA mutations on the contact between promoter DNA and RNAP in the presence of Spx. The same set of experiments shown in Fig. 1 was conducted with Y263C or K267A rpoA mutant RNAPs purified from cells of the spx deletion mutant. Promoter DNA of the trxA, trxB and rpsD genes (A, B and C, respectively) was derivatized with APB at the positions relative to the transcription start site, as indicated by the numbers at the top of each panel. Reactions were performed at 0°C and at 37°C in the presence (WT) and absence (0) of wild-type Spx.

Similar results were obtained in experiments in which reactions contained trxB promoter DNA (Fig. 2B). Incubation of reactions containing RNAPY263C at 37°C resulted in loss of RNAP-DNA promoter contact at positions −14, −34, −46, and −54, as indicated by the absence of cross-linked products. Unlike RNAPY263C, RNAPK267A showed weak σA interaction with the −11 at 37°C, having a σ/α ratio of 1.1 versus 2.4 for wild-type RNAP (determined from data of Fig. 1E). The reduced contact between σ and the −10 region of trxB compared to that of wild-type RNAP is consistent with the observed reduction in transcriptional activity of Spx-RNAPK267A (Zhang et al., 2006). Finally, neither significant loss of cross-linked products nor altered cross-linking pattern between the mutant RNAP holoenzymes and rpsD promoter DNA were detected at 0°C and at 37°C, consistent with previous results (Nakano et al., 2005; Zhang et al., 2006) that these mutations do not significantly affect rpsD transcriptional activity (data not shown).

Identification of a cis-acting element required for Spx-dependent activation

The Spx protein showed no DNA-binding activity in DNase I footprinting experiments (Nakano et al., 2005) or in electrophoretic mobility shift assays with trxA and trxB promoter DNA (D. Y. R. and P. Z., data not shown). Previous studies involving 5′ deletion analyses indicated that sequences downstream from −50 in the trxB promoter and −48 in the trxA promoter were necessary for full transcriptional activation by Spx (Nakano et al., 2005), although these sequences showed no interaction with Spx protein. We sought to further define the sequences upstream of the Spx-controlled promoters required for activation by constructing hybrid promoters composed of the upstream DNA of the trxB promoter and the core promoter DNA from the srf operon (Fig. S3). The srf operon is repressed by Spx-RNAP interaction (Nakano et al., 2003b). Transcription of srf requires the response regulator, ComA, which in its active, phosphorylated form, binds to two upstream palindromic sequences (Fig. S3) to facilitate productive interaction of RNAP with the core promoter elements (Dubnau et al., 1994). Three trxB-srf hybrid promoters consisting of the trxB upstream promoter region and srf core promoter extending up to +47 were constructed (Fig. S3). The hybrid promoters were fused to lacZ and inserted into the thrC locus of a strain bearing an IPTG-inducible spx allele encoding the protease-resistant Spx protein. As controls, the expression of trxB-lacZ and srf-lacZ was examined in the SpxLDD-expressing strain. The control trxB promoter exhibited an 8-fold induction upon SpxLDD overexpression (Fig. 3), whereas no induction was observed for srf-lacZ as expected because Spx-RNAP interaction was previously shown to repress the srf operon (Nakano et al., 2003b). When the −115 to −36 region of trxB promoter was fused with the srf core promoter, SpxLDD overexpression resulted in 4-fold induction of the hybrid promoter activity (Fig. 3B), indicating that the hybrid promoter bears an element required for Spx-dependent transcriptional activation. However, when the trxB-srf hybrid junction was shifted upstream 5 bp to −41, Spx-dependent induction was completely abolished despite SpxLDD overexpression. These results suggested that the 3′-end of a cis-acting element that renders the promoter responsive to Spx is located near nucleotide position −36. Extending the trxB sequence of the hybrid downstream to position −31 resulted in a further increase in the induction ratio (Fig. 3B), suggesting that sequence elements between −36 and −31 also function in Spx-dependent control. This is \ the site of the conserved TAGCGT sequence found in the trxA and trxB promoters (Fig. 1A) (Nakano et al., 2005; Scharf et al., 1998).

Figure 3.

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Promoter activity of trxB-srf hybrid promoters. (A) The effect of SpxLDD production on trxB- (➄) and srf-lacZ (➉) expression. Promoter DNA fused to lacZ was introduced into the thrC locus of B. subtilis cells harboring IPTG-inducible spxLDD. Cultures of each strain were grown in DSM and sampling was carried out every 30 minutes, as described in Experimental Procedures. Time 0 represents time in the growth curve when O.D.600 = 0.3, and 1mM IPTG was added. Activity is expressed in Miller Units (Miller, 1972). (B) Effect of spxLDD expression on levels of β–galactosidase activity of trxB-srf hybrid promoters having fusion joints at −41 (☒), −36 (❶) and −31 (➂). Error bars correspond to standard deviation of three experiments. Induction with IPTG is shown in broken lines while the solid line represents activity in the absence of IPTG.

The interaction of RNAP with srf, trxB and trxB-srf−36 hybrid promoter DNA was examined in the presence and absence of Spx protein. DNase I footprinting reactions containing the 5′-end labeled template strand of each promoter and RNAP were assembled. When increasing concentrations of Spx were added to the reactions, no binding of RNAP to the srf promoter DNA was detected (Fig. 4A). DNase I footprinting showed RNAP interaction with upstream trxB promoter DNA (around −50). This protection is extended into the core promoter region after Spx addition to the reactions (Fig. 4B, lanes marked 0.5 and 1 μM Spx). RNAP in the absence of Spx, protected regions around −50 to −60 of the hybrid trxB-srf −36 promoter from DNase I digestion (Fig. 4C). Addition of Spx enhances protection at regions −10, −30, and −40 of the hybrid promoter. As shown previously (Nakano et al., 2005), the upstream RNAP contacts are due primarily to α subunit-DNA interaction. Thus, similar effects of Spx addition with respect to RNAP-promoter interaction were observed when comparing the footprints of RNAP on hybrid trxB-srf and wildtype trxB promoter substrates. It is important to note that Spx alone (8μM) did not protect promoter DNA from DNase I digestion, as previously observed (Nakano et al., 2005). Two conclusions were drawn from the data of these experiments: The upstream trxB DNA promotes binding of RNAP, and the addition of Spx enhances and extends RNAP binding to the core promoter. This was also reflected in the protein-DNA crosslinking experiments which showed contact of core promoter DNA with σ and ββ′ subunits.

Figure 4.

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Effect of Spx on the ability of RNAP to bind trxB-srf−36 (A), trxB (B), and srf (C), promoter DNA fragments. Radiolabeled template strand was synthesized by 5′-end- labeling the primers using [γ-32P]-ATP and T4 polynucleotide kinase prior to PCR. After incubation with protein, the DNA was then treated with DNase I as described in Experimental Procedures and then subjected to analysis by 8% sequencing gel electrophoresis and phosphorimaging. RNAP holoenzyme (250 nM) and 0, 0.5, 1.0 μM Spx were added to the reactions. Control reactions contained either BSA or 8 μM Spx in the absence of RNAP. The positions relative to the transcriptional start site are indicated, based on the dideoxy sequencing ladders loaded in the left lanes.

Spx enhances cross-linking of RNAP to the hybrid promoter trxB-srf−36, but not the srf promoter

The above results suggest that the fusion of upstream trxB promoter to the srf core promoter provides a site to which RNAP binds, and a cis-acting element required for Spx-dependent transcriptional activation. To test these hypotheses further, cross-linking experiments were undertaken to determine whether similar cross-linking products observed in cross-linking reactions containing trxA and -B promoters are formed in reactions using radiolabeled hybrid promoter. Figure 5A shows that, like trxA and -B promoters, the trxB-srf−36 hybrid promoter exhibits a strong σA cross-linking signal at position −12 of the srf core promoter element of the trxB-srf−36 hybrid at 37°C in the presence of Spx. Mutant Spx (C10A and G52R) did not induce contact between σA and the hybrid promoter as evidenced by the similar cross-linked products as those formed by RNAP and promoter DNA in the absence of Spx. Thus, the Spx requirement for trxB-srf hybrid promoter utilization is the same as that for intact trxB promoter activity. The RNAP large subunits and α showed cross-linking to several positions within and upstream of the srf core promoter in the presence and absence of Spx, again suggestive of non-specific interaction of RNAP with DNA (data not shown). In contrast, RNAP σA, in the presence of wild-type Spx does not contact the srf promoter as indicated by the absence of distinct σA cross-linking product from the −10 motif (Fig. 5B). This is in agreement with the footprinting results wherein no protection of srf promoter DNA by Spx-RNAP was observed. Taken together, these results strongly suggest that the presence of the upstream trxB DNA in the hybrid promoter renders the srf core promoter responsive to Spx-dependent transcriptional activation.

Figure 5.

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Spx enhances binding of RNAP σA to trxB-srf hybrid −36 promoter DNA, but not to srf. Promoter DNA-RNAP cross-linking experiments for trxB-srf hybrid −36 (A) and srf (B) were performed as described for Fig. 1. WT, C and G represent reaction containing 300 nM of wild-type, SpxC10A (C) or SpxG52R (G) mutant Spx, respectively, while 0 indicates a reaction without Spx.

Mutations in the upstream cis-acting element of the trxB promoter abolished Spx-dependent induction

Based on the above result and previous deletion analysis (Nakano et al., 2005), there likely exists a cis-acting element within the −50 to −36 region of trxB that is required for the promoter to be responsive to Spx (Fig. 6A). Sequence alignment of trxA and -B promoters revealed that this might be a conserved element found in promoters directly controlled by Spx. Single point mutations were generated in the upstream element of the trxB promoter that was fused to a promoter-less lacZ gene. The fusion constructs were introduced into the thrC locus of a B. subtilis strain bearing an IPTG-inducible allele of spx, spxLDD (Nakano et al., 2003a). Mutant promoter A-39C showed 2-fold induction whereas A-37C and T-47G exhibited 3 to 4-fold induction (Fig. 6B,C), still less than the 8-fold induction observed with wild-type trxB promoter fused to lacZ. Figure 6C shows that of the mutations introduced into the trxB promoter only C-43A and G-44T substitutions resulted in complete loss of Spx-dependent induction, while A-39C showed only a slight induction upon IPTG addition. These data imply that C-43 and G-44 of the cis-acting element are essential for Spx-dependent induction of trxB transcription initiation.

Figure 6.

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Mutations C-43A and G-44T of trxB confer loss of Spx-dependent induction of trxB transcription. (A) Nucleotide sequence of the regions upstream of the trxA and trxB promoters that contains the putative Spx-responsive cis-acting element. The nucleotide substitutions introduced into the cis-acting element upstream of the trxB promoter are shown. (B) and (C) show the effects of mutations in the region between −37 to −49 of trxB promoter on trxB-lacZ activity. Site-directed mutagenized trxB promoters generated by recombinational PCR were fused to a lacZ reporter gene using plasmid pDG793 (Guerout-Fleury et al., 1996) and introduced to thrC locus of B. subtilis harboring spxLDD linked to an IPTG-inducible promoter. Cells were grown on DSM and β–galactosidase activity was monitored and expressed as Miller Units. IPTG-induced samples are shown in broken line while cells without IPTG-induction are represented as solid line. (B) ORB6826 (WT, ➂), ORB7114 (A-37C, ➄), ORB7122 (T-47G, ➉). ORB7123 (G-49T, ❶). (C) ORB7116 (A-39C, ➄), ORB7118 (C-43A, ❶), ORB7120 (G-44T, ➉), (D) Reduced Spx-dependent transcription activation of mutant trxB promoters in vitro. The indicated amount of Spx was incubated with 25nM RNAP to transcribe rpsD (control) and wild-type (WT) or mutant trxB (C-43A or G-44T) promoters, as described in the Experimental Procedures.

The effects of mutations at C-43 and G-44 on Spx-activated transcription in vitro were examined using trxB promoter DNA as template in run-off transcription reactions containing RNAP and increasing concentrations of Spx (Fig. 6D). The wild-type trxB promoter fragment directed transcription that was responsive to increasing Spx protein concentration. Transcription from the G-44T mutant promoter was much reduced, and activity from the C-43A was nearly undetectable. The transcription data is in agreement with the LacZ assay data, further indicating that the C-43 and G-44 nucleotide positions play an important role in Spx-dependent induction.

The effects on Spx-RNAP interaction with promoter DNA were examined by protein DNA cross-linking (Fig. 7). Wild-type (Fig. 7A), C-43A (Fig. 7B), and G-44T (Fig, 7C) trxB promoter DNA showed non-specific contacts with the α subunit at −4, −10, and −21 positions. The wild-type and, to a lesser extent, the G-44T mutant promoters showed contacts at 37°C between the σA subunit and −11, with σ:α band intensity ratios of 2.1 and 1.4 respectively. Much reduced interaction between σA and −11 is observed in cross-linking reactions containing the trxBC-43A mutant promoter (σ:α ratio of 0.7). The enhanced contact of σ at −11 in the G-44T promoter compared to the C-43A mutant is in keeping with the higher level of in vitro transcript synthesized in the reaction containing trxBG-44T DNA (Fig. 6D). Thus, the data indicates that optimal contact between the σA and the −10 region under conditions that favor open complex formation requires the nucleotides within the cis-acting element residing between −50 and −36.

Fig. 7.

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Effect of mutations in Spx-responsive cis-acting element on binding of RNAP in the presence of Spx. Protein-DNA cross-linking experiments were performed as in Fig. 1B using APB-derivatized fragments of trxB promoter DNA (A) and those of the C-43A (B) and G-44T (C) derivatives. Nucleotide positions −4, −11, and −24 were modified with APB and reactions contained 25 nM of RNAP and 300 nM of Spx where indicated.

Discussion

The mechanism of Spx-dependent transcriptional activation is unknown at present, due in part to the fact that Spx is structurally unlike any other transcription factor and that it shows no sequence-specific DNA binding activity (Nakano et al., 2005; Zuber, 2004). It is known that it can interact with the α subunit of RNAP, and an interaction surface involving the helix 1 of the RNAP α subunit C-terminal domain has been identified (Newberry et al., 2005). While this finding provided a model for how Spx exerts negative control, by interfering with activator-RNAP interaction (Zhang et al., 2006), it did not provide sufficient information of how Spx-dependent activation at specific promoter sequences could operate. The oxidized, disulfide form of Spx is also necessary for activation of trxB and trxA transcription, but how this alters the RNAP-promoter interaction so as to induce transcription initiation is not known. The apparent ability of Spx to alter the architecture of promoter bound-RNAP without engaging in nucleotide-specific DNA interactions suggests that Spx acts as an appropriator (Campbell et al., 2008) by changing promoter specificity of the holoenzyme.

The present study provides some clues to how Spx activates transcription and has uncovered the requirement for a cis-acting element functioning in transcriptional activation that is likely shared by trxB and trxA promoters. Similar sequences can be found upstream of putative −35 regions of σA promoters in other genes induced by Spx-RNAP interaction, such as yugJ, (CAGCACCTT), yraA (GAGCAAATA), and yqjM (GAGCAGTTTA). The nfrA gene, which encodes a FMN-containing NADPH-linked nitro/flavin reductase (Zenno et al., 1998), is highly induced in cells overexpressing Spx (Nakano et al., 2003a). A putative σA promoter was identified in the nfrA gene, and sequences within were found to be required for nfrA transcription (Moch et al., 2000). An upstream sequence (−46 GAGCATTTCA −38) in the nfrA promoter region resembles the cis-acting element found upstream of trxA and trxB. The G-44T and the C-43A mutations in the upstream sequence of the nfrA promoter were analyzed by Moch et al. (Moch et al., 2000) and were shown to cause a 10–20-fold reduction in nfrA transcription. These two positions correspond to the GC nucleotides at −44 and −43 that are required for Spx-dependent activation of trxB transcription.

The fusion of the trxB upstream sequence, 5′ to the −35 region, to the core promoter of srf yields a hybrid promoter to which B. subtilis RNAP binds in the presence of Spx. While protein DNA cross-linking showed non-specific contacts between RNAP and the srf promoter, DNase I footprinting analysis shows no interaction between RNAP and srf DNA. This is in keeping with the fact that this interaction requires the activated form of the response regulator, ComA (Zhang et al., 2006). Addition of Spx did not change the pattern of RNAP-srf DNA cross-linking nor did it promote any detectable RNAP-srf interaction based on the footprinting data. Based on these observations, the cross-linking of ββ′ and α to the srf promoter DNA is likely weak nonspecific RNAP-DNA interaction. RNAP protected the upstream, trxB region of the trxB-srf−36 hybrid construct to around −50, as well as nucleotide positions within the core srf promoter, suggesting that contact of RNAP with DNA upstream of the core promoter is required for productive Spx contact with the transcription complex, resulting in σ and ββ′ interaction with downstream core promoter elements. Interaction of RNAP with the upstream regions of trxB and trxA promoter DNA is mediated, in part, by the α subunit (Nakano et al., 2005). Interaction of RNAP α subunit with upstream DNA of certain promoters involves multiple RNAP-DNA contacts that can lead to compaction of DNA and wrapping of the polydeoxynucleotide chains around the bound holoenzyme (Cellai et al., 2007). This can increase the affinity of RNAP for promoter DNA and also generate distortion of the helical environment, thus promoting open complex formation (Aiyar et al., 1998; Naryshkin et al., 2000; Ross and Gourse, 2005). Nucleotide sequence- independent interactions in the upstream DNA by the RNAP α subunit affects binding of RNAP to promoter DNA and isomerization at many promoters (Ross and Gourse, 2005). The nonspecific binding of α to DNA, which is destabilized by the rpoAY263C and rpoAK267A mutations at 37°C (Fig. 2), might be required for optimal Spx-dependent activation. These mutations also render defective the productive interaction between Spx and RNAP (Nakano et al., 2005; Zhang et al., 2006). The relationship between α and Spx is similar to the interaction between SoxS and RNAP, which involves residues of the α subunit that normally function in DNA binding (Shah and Wolf, 2004). Therefore, requirements for Spx-activated transcription might include the sequence independent interaction of the α subunit with promoter DNA and Spx/α complex interaction with the cis-element centered at −44 and upstream of the core promoter elements.

Cross-linking experiments uncovered changes to the organization of the transcription complex occuring at 37°C. Enhancement of σ subunit binding only to the trxA promoter is observed at 0°C when Spx is included in the reaction. Unlike trxB, the trxA promoter contains an extended −10 region bearing a TG motif, which plays a role in RNAP binding to promoter DNA and renders the RNAP-promoter complex less dependent on σ interaction with the −35 sequence (Burr et al., 2000; Camacho and Salas, 1999). That Spx enhances σ subunit contact with the trxA −10 region at 0°C suggests that Spx can affect the binding step of RNAP-promoter interaction. At 37°C, Spx interaction with promoter-bound RNAP could result in alteration of RNAP-promoter architecture to induce further σA and the ββ′ subunit engagement with the −10 region and nucleotide positions near the start site. One possible effect of Spx interaction is the repositioning of the σ subunit in the −35 region, which then allows proper interaction of σ region 2 with the −10 sequence. Such a mechanism of activation has been proposed for transcription initiation stimulated by the response regulator, Spo0A in B. subtilis (Kumar and Moran Jr, 2008; Seredick and Spiegelman, 2004). Our cross-linking data did not show contact between the σ subunit and sequences in the −35 region or in the cis-acting element centered at −43 and −44, however. Nor could we detect contact of Spx with trxA and trxB promoter DNA. It is possible that modifying the target DNA with APB for cross-linking blocks interaction with Spx and/or RNAP subunits. This could be the explanation for why contact between the σ subunit and DNA in the −35 region was not detected using UV-activated cross-linking. Mutations in region 4.2 of the σA subunit can impair Spx-dependent activation (Y. Zhang and P. Z., unpublished), but no interaction between Spx and σA has been detected (K. Newberry and R. G. Brennan, personal communication). Nevertheless, the results of σA mutational analysis suggests that σA might play an additional role aside from engaging the −10 region during Spx-dependent activation. The sequence TAGCGT is found in the −35 regions of both trxA and trxB as noted previously (Nakano et al., 2005), and was proposed to be the −35 sequence of the trxA promoter (Scharf et al., 1998), although it possesses only 2 of the 6 nucleotides of the consensus −35 sequence of σA-utilized promoters. The trxB-srf−31 hybrid contains 5 of the six nucleotides of this motif (TAGCG) and shows a higher level of Spx-dependent induction than the trxB-srf−36 hybrid (Fig. 3). Inclusion of this sequence in the trxB-srf hybrid promoter did not result in an increase in basal level trxB-srf-lacZ expression (Fig. 3, -IPTG), suggesting that the increased induction is not due to an enhancement of the −35 consensus sequence in this region. The sequence is not conserved among other Spx-induced genes. At this time, the function of the TAGCGT sequence in Spx-dependent control is unclear.

Experimental Procedures

Bacterial strains and media

All B. subtilis strains listed in Supplemental Table S1 are derivatives of JH642. Strains ORB6824, 6827, 6881, 6979, 7113, 7115, 7117, 7119 and 7121 were generated by transforming JH642 through integration of pDYR9, pDYR10, pDYR16, pDYR23, pDYR24, pDYR25, pDYR26, pDYR27 and pDYR28, respectively, at the thrC locus by double-cross-over recombination and screening for threonine auxotroph phenotype. ORB4342 was transformed using the same set of plasmids to construct strains ORB6826, 6829, 6883, 6981, 7114, 7116, 7118, 7120 and 7122, respectively.

B. subtilis cells were grown in Difco sporulation medium (DSM), and antibiotics were added to the following final concentrations: erythromycin plus lincomycin, 1 and 25 μg ml−1; spectinomycin, 75 μg ml−1. Escherichia coli cells were grown in either 2X Yeast extract, tryptone (2XYT) or LB medium with ampicillin added to a final concentration of 50μg ml−1.

Plasmid constructions

To construct plasmid pDYR9, an insert DNA bearing the full-length trxB promoter (−115 to +47) was amplified by PCR from JH642 chromosomal DNA using primers oSN03-78 and oDYR06-032 and first introduced into SmaI-HindIII-cleaved pTKlac to generate pDYR5. Insert DNA of pDYR10 (hybrid −36) and 16 (hybrid −41) were generated as follows: primary PCR products were first amplified using the common primer set oSN03-78 and oYZ02-4 in combination with oDYR06-030 and oDYR06-029-2 for hybrid −36 and oDYR07-039 and oDYR07-038 for hybrid −41. pDYR5 was used as template DNA. Hybrid promoters were then generated by recombinant PCR using primers oSN03-78 and oYZ02-4. The resulting PCR fragments were digested with SmaI and HindIII, and then inserted into pTKlac or pUC18 to generate pDYR6 (hybrid −36) and pDYR12 (hybrid −41), respectively. DNA promoters were excised from plasmids pDYR5, 6 and 12 via EcoRI and HindIII digestion and insertion into the same sites of plasmid pDG793 to obtain plasmids pDYR9, 10 and 16, respectively. Likewise, srf promoter (−184 to +48) DNA was PCR-amplified using primers oDYR07-049 and oYZ02-4, digested with EcoRI and HindIII, and inserted into pDG793 to generated pDYR23.

Plasmids bearing single point mutation in the trxB promoter were constructed as follows: primary PCR products were first amplified using the common outside primers oDYR07-052 and oDYR06-032 in combination with oDYR07-053-oDYR07-054, oDYR07-055-oDYR07-056, oDYR07-057-oDYR07-058, oDYR07-059-oDYR07-060, oDYR07-061-oDYR07-062 or oDYR07-063-oDYR07-064 to generate transversion mutations A-37C, A-39C, C-43A, G-44T and T-47G, respectively. These fragments were used as templates to generate recombinant PCR products using the aforementioned common primers, then followed by digestion with EcoRI and HindIII. The restriction cleaved fragements were inserted into pDG793 to obtain plasmids pDYR24, 25, 26, 27 and 28, respectively. Sequences of oligonucleotides used in plasmid construction and in vitro transcription are avaible upon request.

Promoter DNA-Protein Crosslinking

Crosslinking probes were synthesized according to the method described by Bartlett, et al. (Bartlett et al., 2000) with minor modifications. First, strepavidin-bound DNA promoter fragments were generated by PCR and purified using low melting agarose gel electrophoresis. Twenty pmol of oligonucleotide containing a single, 3′ phosphorothioate substitution was then annealed with an equal concentration of purified strepavidin-bound DNA template and adjacently radiolabeled with 120 μCi [α-32P]-dATP (MP Biomedicals) using Klenow (exo−) enzyme. Full-length extension product was \ synthesized after addition of T4 DNA polymerase and dNTP mix (10 mM each). To remove the biotin group, the radiolabeled DNA was treated with HaeIII (New England Biolabs) for 3 hours then subsequently extracted with phenol-chloroform-isoamyl alcohol (PCI) solution and precipitated with ethanol. After drying, probes were subjected to derivatization with crosslinking by treating overnight with 100 mM azidophenacyl bromide (APB) in the presence of 100 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.0) in the dark at room temperature. The derivatized products were then PCI-extracted, ethanol precipitated, and the precipitate was collected by centrifugation before finally dissolving derivatized probe DNA in distilled water.

For the crosslinking experiments, wild-type or mutant RNAP with or without Spx were suspended in buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5% glycerol and 100 μg ml−1 BSA on ice. In the dark, open (37°C) or closed (on ice, or 4°C) complex formation was initiated after addition of radiolabeled probes (10000 cpm) and incubation for 30 mins. Crosslinking was then carried out for 10 seconds using a UV Stratalinker 1800 (Stratagene), after which samples were immediately put on ice. Samples were subjected to DNase I and S1 nuclease digestion at 37°C for 10 mins each before finally resolving the crosslinking products by electrophoresis through a 12% SDS-PAGE gel. Imaging of gel profiles was done using a Typoon Trio+ Variable Mode Imager. Quantification of band intensities was accomplished using ImageJ software on multiple gel images.

In vitro run-off transcription assay

Linear rpsD and trxB promoter DNA templates were generated through PCR with primers oSN03-86 and 0SN03-87 (encoding a 71-base transcript), and oDYR07-032 and oDYR07-052 (encoding a 66-base transcript), respectively. The wild-type trxB promoter was amplified from pDYR9, while mutant promoters trxBC-43A and trxBG-44T were synthesized from pDYR26 and pDYR27, respectively. The templates were mixed in the reaction to the following final concentrations: 10nM with trxB and 20nM with rpsD. In vitro transcription experiment was performed as follows: RNAP and template DNA were incubated with or without Spx in buffer containing 10mM Tris-HCl (pH 8.0), 50mM NaCl, 5mM MgCl2 and 50 μg ml−1 BSA for 10 minutes at room temperature. Reaction is then initiated after addition of nucleotide mixture (200 μM each of ATP, GTP and CTP, 10 μM UTP, 10 μCi [α-32P]-UTP) at 37°C, and stopped after 15 minutes by adding a 10 μl solution containing 1 M ammonium acetate, 30 mM EDTA and 0.1 mg ml−1 yeast tRNA. Transcripts were ethanol precipitated and resolved using an 8% Urea-PAGE gel. Images were scanned using a Typhoon Trio+ (Amersham Biosciences) and processed using Image Quant 5.2.

DNase I footprinting

DNA probes for trxB (179 bp), srf (248 bp) and trxB-srf−36 (179 bp) hybrid promoters were made by PCR amplification with primers oSN03-78 and oSN03-61, oDYR07-049 and oYZ02-4, and oSN03-78 and oYZ02-4, respectively. Plasmids pSN78 (trxB), pDYR23 (srf) and pDYR10 (trxB-srf−36) were used for the PCR templates. Radiolabled DNA substrate was synthesized by end-labeling one member of each of a PCR primer set using T4 polynucleotide kinase and [γ-32P]-ATP prior to PCR. PCR product was then resolved in a 6% non-denaturing gel and purified using an Elutip-D column (Schleicher and Shuell). The thermo Sequenase Cycle Sequencing Kit (USB) was used to synthesize the sequencing ladder for the footprinting reactions. DNase I footprinting experiments were carried out as follows: 50,000 cpm of radiolabeled probe was added to RNAP with or without Spx in a buffer consisting of 10 mM Tris-HCl (pH 8.0), 30 mM KCl, 10 mM MgCl2 and 100 μg ml−1 BSA at 37°C for 20 minutes. After another incubation at room temperature for 5 minutes, the reaction mixture was treated with 15 mU of DNase I for 10 seconds. Reactions were stopped with phenol-chloroform-isoamyl alcohol solution then ethanol precipitated. Electrophoresis was carried out in 8% denaturing PAGE gel.

Protein purification

To generate strains harboring His10-rpoC in an spx deletion background, strains MH5636, ORB4123 and 6116 were transformed with chromosomal DNA derived from ORB3834 to obtain ORB4028, 6471 and 6499, respectively. RNAP was then stepwise purified from these strains from 4 L of 2XYT cultures. Cells were lysed by three passages through a French press, and cleared lysate was collected by centrifugation using a procedure described previously (Liu and Zuber, 2000). RNAP was obtained by first elution from a Ni-nitrilotriacetic acid (Ni-NTA, Qiagen) column. The eluted RNAP was applied to a heparin-agarose column and eluted with a 100–800 mM NaCl gradient, then further purified by applying the preparation onto a Bio-Rad High Q column, followed by elution with a 100–500 mM NaCl gradient, as previously described (Choi et al., 2006). Finally, purified RNAP was concentrated with a Centricon YM-10 (Millipore) concentrator, then dialyzed against 10 mM Tris-HCl (pH 8.0), 100 mM KCl, 10 mM MgCl2 and 50% glycerol and stored at −20°C.

Intein-tagged Spx proteins were purified according to the procedure described previously (Nakano et al., 2002) with minor modifications. E. coli ER2566 cells harboring the plasmids encoding the wild type or mutant spx alleles were grown in 2XYT with ampicillin. At O.D.600=0.5, IPTG was added to final concentration of 0.5 mM and cells were allowed to grow for another 5 hours. Harvested cells were passed three times through a French press and centrifuged at 14,000 rpm at 4°C for 30 minutes to collect the supernatant. Spx proteins were then loaded onto and then eluted from a chitin column (New England Biolabs), according to the manufacturer’s recommendation. Spx was further purified by Bio-Rad High S column chromatography. Purified proteins were concentrated and dialyzed against 25 mM sodium phosphate (pH 6.8), 100 mM KCl and 5% glycerol and stored at −80°C.

Assay for β–galactosidase activity

Strains harboring trxB-lacZ, srf-lacZ or trxB-srf-lacZ fusions were grown DSM and samples were withdrawn on 30-min interval during growth. For samples subjected to Spx overexpression, IPTG was added to final concentration of 1 mM after O.D.600=0.3. β–galactosidase activity was measured as described and is presented as Miller unit (Miller, 1972;Zuber and Losick, 1983).

Supplementary Material

Fig.1S-3S
supplement 1

Acknowledgments

The authors wish to thank M. Bartlett and K. Erwin for technical advise on cross-linking, and M. M. Nakano for helpful advice and critical reading of the manuscript. Research reported herein was funded by grant GM45898 from the National Institutes of Health, USA.

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Supplementary Materials

Fig.1S-3S
NIHMS65534-supplement-Fig_1S-3S.pdf (1.3MB, pdf)
supplement 1
NIHMS65534-supplement-supplement_1.pdf (227.3KB, pdf)

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