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Journal of Bacteriology, March 2007, p. 1736-1744, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01520-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dual Negative Control of spx Transcription Initiation from the P₃ Promoter by Repressors PerR and YodB in Bacillus subtilis

Montira Leelakriangsak,¹ Kazuo Kobayashi,² and Peter Zuber^1*

Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon,¹ Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan²

Received 28 September 2006/ Accepted 4 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P₃ promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P₃ promoter. Expression of an spx-bgaB fusion carrying the P₃ promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P₃ transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P₃ promoter DNA was used as a template. Point mutations at positions within the P₃ promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P₃ –10 and –35 regions, while PerR binds to a region downstream of the P₃ transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Spx is a highly conserved transcriptional regulatory protein of low-GC-content gram-positive bacteria (2, 4, 7, 29, 36, 40). It does not possess sequence-specific DNA-binding activity but instead directly targets RNA polymerase (RNAP) (26, 28), an interaction that interferes with contact between transcriptional activators (26, 39) and RNAP while activating transcription at promoters of genes whose products function in intracellular thiol homeostasis (24, 25) and responses to encounters with toxic oxidants (25, 29, 33, 34). Recent studies suggested that Spx plays a role in cellular invasion by a microbial pathogen (2). Spx does not belong to any other known family of transcriptional regulatory proteins but resembles the arsenate reductase ArsC of plasmid R773 in terms of its primary and higher-order structure (18, 28, 40).

The spx gene resides in the yjbC-spx operon of the Bacillus subtilis genome and is transcribed from at least five promoters by four forms of RNAP holoenzyme (^A, ^B, ^M, and ^W). Induction of spx expression has been associated with phosphate starvation, ethanol, and oxidative stress (1, 25, 35), while induction of the spx regulon is activated by a variety of stress conditions that include heat shock, salt stress, oxidative stress, and toxic phenolic compounds (25, 33, 34). The control of spx expression in response to oxidative stress operates at three levels. Spx protein levels increase independently of spx transcriptional control, perhaps by the downregulation of ClpXP-catalyzed proteolysis (25, 27). Spx activity is under redox control through its CxxC disulfide center (24), and, finally, transcription from the newly discovered P₃ promoter of spx is induced by disulfide stress, as shown in the accompanying paper (15a). The tight control of spx expression and the activity of its product likely provide assurance that Spx is not overproduced inappropriately, since high concentrations of Spx have a severe effect on growth and a variety of transition-state developmental processes (21, 23, 25, 26).

The analysis of the P₃ promoter region of spx uncovered two negatively cis-acting elements, mutations of which cause the derepression of spx transcription under normal growth conditions. In this report, we show that the two sites participate in the repression of spx transcription that is exerted by the direct interaction of two negative transcriptional regulators, PerR and YodB. PerR is the previously characterized peroxide stimulon control factor (13, 15), while YodB is a novel DUF24 family member that exerts repression that is sensitive to treatment with oxidants.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions. Bacillus subtilis strains used in this study are derivatives of JH642 and are listed in Table 1. Cells were cultivated in a shaking water bath at 37°C in Difco sporulation medium (DSM) (31) for ß-galactosidase assays or TSS minimal medium (6) for diamide treatment experiments. Diamide was purchased from Sigma. To create ORB6118, the strain used in the transformation array experiment, the following constructions were carried out. Plasmid pSN16 (22) was digested with EcoRI to release the fragment containing only the spx promoter (spx coding sequencing including 538 bp upstream of P₃) to generate pML8 (spx-lacZ fusion). pML8 was transformed into ZB307A (41) with selection of chloramphenicol resistance. SPß-transducing lysate was produced by heat induction and was then transduced into ZB278 (41). The phage generated from this strain was used to transfer the spx-lacZ fusion into the wild-type (wt) background by transduction into JH642 with selection for chloramphenicol resistance to generate strain ORB4563. Plasmid pCm::Sp (32) was used to transform ORB4563, replacing the Cm^r cassette with an Spc^r cassette to generate strain ORB6118.

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TABLE 1. Bacillus subtilis strains and plasmids used in this study

The isogenic yodB and perR mutants were constructed by transformation of JH642 with chromosomal DNA of TF277 (this study) and HB2078 (8), respectively. The resulting strains were designated ORB6208 (yodB::cat) and ORB6267 (perR::kan).

The yodB mutant (TF277) was constructed as follows. Upstream and downstream regions of yodB were amplified by PCR with the primer sets yodB-F1/yodB-R1 and yodB-F2/yodB-R2 (Table 2), respectively. The cat (chloramphenicol acetyltransferase) gene was amplified by PCR from plasmid pCBB31 using primers PUC-F and PUC-R. The 5' ends of yodB-F2 and yodB-R1 are complementary to the sequence of PUC-F and PUC-R, respectively. Three PCR products were mixed and used as templates for the second PCR with primers yodB-F1 and yodB-R2 (Table 2). The resultant PCR fragment amplified via overlap extension was used for the transformation of B. subtilis 168.

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TABLE 2. Oligonucleotides

The fragment containing the spx P₃ promoter region (positions –330 to +50 relative to the P₃ transcription start site) was fused with the promoterless bgaB gene (plasmid pDL) (37) as the reporter that was integrated into the amyE locus of JH642 to generate strain ORB5058 [spxP₃+50(wt)-bgaB] (15a). Plasmid pCm::Tc (32) was used to transform ORB5058 competent cells, thereby replacing the cat cassette with a tet (tetracycline resistance) cassette to generate strain ORB6284. For perR, yodB, and perR yodB disruption strains bearing the spx-bgaB fusion, chromosomal DNA of ORB6208 (yodB::cat) was transformed into ORB6284 to generate ORB6288 [spxP₃+50(wt)-bgaB yodB::cat]. Chromosomal DNA of ORB6267 (perR::kan) was used to transform cells of strain ORB5058 to generate ORB6268 [spxP₃+50 (wt)-bgaB perR::kan]. The yodB perR double mutant was constructed by transformation of ORB6288 with chromosomal DNA from ORB6267 to generate ORB6594 [spxP₃+50(wt)-bgaB yodB::cat perR::kan].

For the complementation experiment with yodB, primers oyodB-EcoRI and oyodB-HisB (Table 2) were used to amplify the yodB gene from B. subtilis strain JH642 chromosomal DNA. The PCR fragment (about 550 bp, including the coding region of yodB as well as 200 bp of upstream sequence and 15 bp of downstream sequence) was digested with EcoRI and BamHI and ligated with pUC19 digested with the same enzymes to generate pML63. The yodB sequence in plasmid pML63 was verified by DNA sequencing. pML63 was cleaved with EcoRI and BamHI, and the released yodB fragment was inserted into pDG795 (9), which was digested with the same enzymes, to generate pML64. pML64 was introduced by transformation, with selection for erythromycin-lincomysin, into B. subtilis strain JH642, where the yodB fragment integrated into the thrC locus. The resulting strain was designated ORB6606 (thrC::yodB). Chromosomal DNA of ORB6606 was used to transform ORB6288 to generate ORB6616 [spxP₃+50(wt)-bgaB yodB::cat thrC::yodB]. Cells were grown in DSM until the optical density at 600 nm (OD₆₀₀) reached 0.4 to 0.5. The cells were harvested and prepared for ß-galactosidase assays after further incubation for 30, 60, and 120 min.

Double mutants bearing spx-bgaB fusions with cis-acting mutations and yodB or perR mutations were constructed as follows. Plasmid pCm:Sp was used to transform ORB6208 competent cells, thereby replacing the cat cassette with an spc cassette to generate strain ORB6299. Chromosomal DNA of ORB6267 (perR::kan) or ORB6299 (yodB::spc) was used to transform ORB6030 (T–26A), ORB6031 (T–20G), ORB6032 (T–19G), ORB6033 (A–14T), ORB6034 (A3G), ORB6035 (T7C), and ORB6036 (T24C) to generate ORB6684 (T–26A yodB::spc), ORB6685 (T–26A perR::kan), ORB6686 (T–20G yodB::spc), ORB6687 (T–20G perR::kan), ORB6688(T–19G yodB::spc), ORB6689(T–19G perR::kan), ORB6690 (A–14T yodB::spc), ORB6691 (A–14T perR::kan), ORB6692 (A3G yodB::spc), ORB6693 (A3G perR::kan), ORB6694 (T7C yodB::spc), ORB6695 (T7C perR::kan), ORB6696 (T24C yodB::spc), and ORB6697 (T24C perR::kan).

Transcription factor array analysis. The transcription factor/transformation array analysis was performed as previously described (11). Competent cells of strain ORB6118 bearing the prophage SPßc2del::Tn917::pML8-15 (spxP₃-lacZ) were used for the transformation array.

Protein purification. The yodB coding sequences was amplified by PCR using primers oyodB-HisN and oyodB-HisB (Table 2). The PCR products were digested with NdeI and BamHI restriction enzymes and inserted into pPROEX-1 (Life Technologies) digested with the same enzymes to generate pML54. Escherichia coli M15 cells carrying pRep4 and pHis-PerR (12) were cultured in 100 ml LB medium, or BL21 (pML54) was cultured in 500 ml LB medium, and IPTG (isopropyl-ß-D-thiogalactopyranoside) (final concentration, 0.5 mM) was added at the mid-log phase (OD₆₀₀ of 0.6). After 5 h, the cells were harvested, collected by centrifugation, and resuspended in lysis buffer A (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole) with 1 mM phenylmethylsulfonyl fluoride. The cells were disrupted using a French pressure cell and centrifuged. An equal volume of 50% Ni-nitrilotriacetic acid (NTA) resin (QIAGEN) was added to the lysate, mixed into the column, and shaken at 4°C for 3 h. The column was washed with wash buffer B (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole). His₆-PerR or His₆-YodB was eluted with elution buffer C (50 mM NaH₂PO₄, 300 mM NaCl, 200 mM imidazole). PerR or YodB eluted from the Ni-NTA column was further purified by High Q column chromatography (Bio-Rad) with a 50 to 500 mM NaCl gradient. One milligram of His-tagged YodB was incubated with 500 U AcTEV (Invitrogen) at 30°C for 4 h to remove the N-terminal His₆ tag. The AcTEV protease was removed from the cleavage reaction mixture by Ni-NTA resin followed by elution of YodB with elution buffer containing 20 to 40 mM imidazole. The purified PerR and cleaved YodB were extensively dialyzed against TEDG buffer (50 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 2 mM dithiothreitol, and 10% glycerol) and stored at –80°C.

In vitro transcription assay. Linear DNA templates for rpsD and spx promoters were generated by PCR with primers oSN86 and oSN87 (24) (encoding a 71-base transcript) and with oML02-37 and oML02-22 (encoding a 50-base transcript) (15a), respectively. Linear DNA templates for spx point mutation promoters were generated by PCR with primers oML02-37 and oMLbgaB encoding a 110-base transcript using pML42, pML43, pML44, and pML46 as templates for T–26A, T–20G, T–19G, and A–14T spx mutations, respectively. Primers oML02-37 and oML02-22 (15a) were used to generate T+24C linear DNA templates using pML48 as a template encoding a 50-base transcript. The 3' spx promoter deletion DNA templates were also generated by PCR with primers oML02-37 and oMLbgaB (15a) encoding a 100-base transcript for position +40 and a 65-base transcript for position +5 using pML34 and pML30 as templates, respectively. The 20 nM DNA templates were mixed with 50 nM RNAP and ^A (20) and then incubated with and without PerR or YodB in a solution containing 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 50 µg/ml bovine serum albumin, and 5 mM dithiothreitol at 37°C for 10 min. A nucleotide mixture (200 µM ATP, GTP, and CTP, 10 µM UTP, and 10 µCi [-³²P]UTP) was added to the reaction mixture. The reaction mixtures (20 µl) were further incubated at 37°C for 15 min, and the transcripts were precipitated by ethanol. Electrophoresis was performed as described previously (17).

DNase I footprinting. DNA probes for spx (position corresponding to positions –100 to +70), spx(T–19G), spx(T–26A), and spx(T+24C) (positions –100 to +50) were made by PCR amplification using primers oML02-15 and oML02-25 and primers oML02-25 and oML02-22 (15a), respectively. Plasmids pSN16 (spx wild-type promoter) (27), pML42 [spx(T–19G)], pML44 [spx(T–26A)], and pML48 [spx(T+24C)] (15a) were used as PCR templates. The DNA probe for yrrT was made as described previously (3). The coding-strand primer (oML02-25) (15a) was treated with T4 polynucleotide kinase and [-³²P]ATP before the PCRs. The PCR products were separated on a nondenaturing polyacrylamide gel and purified with Elutip-d columns (Schleicher & Schuell). Dideoxy sequencing ladders were obtained using the Thermo Sequenase cycle sequencing kit (USB) and the same primer used for the footprinting reactions. DNase I footprinting reactions were performed using a solution containing 10 mM Tris-HCl (pH 8.0), 30 mM KCl, 10 mM MgCl₂, and 0.5 mM ß-mercaptoethanol. Diamide (1 mM and 1.5 mM) was used to detect the effect of diamide on the DNA binding. The concentrations of H₂O₂ used were 25 mM, 50 mM, and 100 mM to detect the effect of H₂O₂ on the DNA binding. Proteins were incubated with a 100,000-cpm-labeled probe at 37°C for 20 min prior to DNase I treatment. The reaction mixtures were then precipitated by ethanol and subjected to 8% polyacrylamide-8 M urea gel electrophoresis.

Primer extension analysis. Strains JH642 (wild type), ORB6208 (yodB::cat), ORB6267 (perR::kan), and ORB6324 (yodB::cat perR::kan) were grown at 37°C in TSS medium. Primer extension analysis was performed as described in the accompanying paper (15a).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two genes that negatively control spx transcription. In the accompanying paper, the discovery of the spx P₃ promoter and its activation during disulfide stress are described (15a). P₃ resides in the intergenic region between yjbC and spx, 79 bp upstream from the spx ATG start codon. It is utilized by the ^A form of RNA polymerase and is the only transcriptional start site detected in the yjbC-spx intergenic region when cells are grown under the conditions used in our studies. Mutational analysis has uncovered two potential cis-acting negative control elements for P₃-directed transcription initiation, one downstream of the transcriptional start site and the other within the P₃ promoter itself.

Four different forms of RNA polymerase (^A, ^B, ^W, and ^M) contribute to spx transcription, and there has been one previously published report of a negative regulatory factor, PerR, that controls spx expression (12). We sought to uncover other transcription factors that exert negative control on transcription from P₃ using transcription factor/transformation array technology (11). A lacZ fusion of the yjbC-spx intergenic region extending from position –538 to the spx stop codon was constructed and screened for increased ß-galactosidase activity in the transcription factor mutant backgrounds. Figure 1 shows a portion of the array in which a colony of the fusion-bearing strain transformed with yodB::cat DNA appears slightly more blue (darker in the black-and-white image) (Fig. 1) on the X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) DSM agar plate than neighboring transformant colonies.

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FIG. 1. Transcription factor transformation array using an spx-lacZ fusion strain as a recipient. Colonies on the left are transformants of a strain that were transformed with DNA from individual mutants with an insertion in genes encoding known or putative transcription factors. The genes mutated are indicated on the right and correspond to the pattern of colonies on the left.

To validate the transformation array results and previously reported microarray data, the spx-bgaB fusion was introduced into a perR and a yodB drug resistance insertion mutant, and fusion expression was examined. Expression of spx-bgaB was constant throughout growth and into the stationary phase in wild-type B. subtilis cells (Fig. 2A). In perR mutant strain ORB6268, spx-bgaB activity increased significantly over that of wild-type cells. A lower level of derepression was observed in the yodB mutant, and an additive effect of the absence of yodB and perR was observed in the double mutant. Figure 2B shows the activity of spx-bgaB in a yodB mutant strain, ORB6288, that bears an ectopically expressed wild-type copy of yodB, which confirms that the absence of YodB in the yodB insertion mutant is the cause of spx-bgaB derepression.

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FIG. 2. Effect of the disruption of perR, yodB, and perR yodB on the expression of spx-bgaB cells. The cells were grown in DSM, and their ß-galactosidase (BgaB) activities were determined as described in Materials and Methods. Time zero indicates the mid-log phase. Triplicate experiments were performed. •, ORB6284 (wild type); , ORB6288 (yodB mutant); , ORB6268 (perR mutant); x, ORB6594 (yodB perR double mutant). (B) Complementation experiment of yodB. ß-Galactosidase activities of strains containing spx-bgaB and yodB::cat complemented with yodB are depicted. Cells were grown in DSM. Samples were taken after the OD600 reached 0.4 to 0.5 (time zero) and at 30 min, 60 min, 120 min, and 180 min. Results are means ± standard deviations from three independent experiments. •, ORB6284 (wild type); , ORB6288 (yodB::cat); , ORB6616 (yodB::cat thrC::yodB).

Primer extension analysis to determine the level of P₃ transcript in JH642 cells as well as in cells of the yodB mutant derivative (ORB6208), the perR mutant (ORB6267), and the yodB perR double mutant (ORB6324) was performed. As was shown previously, diamide treatment increased spx P₃ transcript levels (Fig. 3, lanes 1 to 3). An increase in P₃ transcript was also observed in the diamide-treated yodB (Fig. 3, lanes 4 to 6) and perR (lanes 7 to 9) mutant cells. Importantly, the P₃ transcript concentration was high in untreated yodB and perR mutants compared to untreated wild-type cells (Fig. 3, compare lanes 1 and 2 to 4 and 5 and 7 and 8), which is indicative of the roles that the two regulators play in the negative control. A further increase was observed in the untreated cells of the yodB perR double mutant (Fig. 3, lanes 10 and 11), as was predicted from the BgaB assay data shown in Fig. 2, which showed a level of transcriptional activity that was higher than the activity of either the yodB or perR single mutant. Unexpectedly, when treated with diamide, the double mutant reproducibly showed sharply reduced levels of spx P₃ transcript (Fig. 3, lane 12).

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FIG. 3. Primer extension analysis of RNA extracted from JH642, ORB6208 (yodB mutant), ORB6267 (perR mutant), and ORB6324 (yodB perR double mutant) cells in cultures subjected to diamide treatment. Cells were treated with 1 mM diamide for 10 min (10D) and without diamide (0 and 10 min) after the OD600 reached 0.4 to 0.5. Labeled primer oML02-15 was used for the primer extension reaction. The dideoxy sequencing ladders are shown on the left. For dideoxynucleotide sequencing, the nucleotide complementary to the dideoxynucleotide added in each reaction mixture is indicated above the corresponding lane (T', A', C', and G').

Purified PerR and YodB proteins repress transcription from spx P₃ in vitro. YodB and PerR were obtained from E. coli expression systems in a His₆-tagged form for use in transcription reactions and protein-DNA-binding experiments. Both proteins were purified from cleared lysates by Ni-chelate and ion-exchange chromatography. The PerR protein was used in the His-tagged form (12), while His₆-taggedYodB was proteolytically cleaved to release the N-terminal His tag sequence (see Materials and Methods).

The purified YodB and PerR proteins were added to in vitro runoff transcription reaction mixtures containing purified RNAP and ^A from B. subtilis and linear spx P₃ DNA fragments generated by PCR. Template DNA was also obtained by PCR amplification of mutant P₃ promoter DNA that was isolated as described in the accompanying paper (15a). The spx mutations T24C, T–26A, T–20G, T–19G, and A–14T caused spx transcription from P₃ to be higher than that observed in wild-type cells (15a). Deletions of the region 3' to the P₃ transcription start site also resulted in the derepression of transcription from P₃. The addition of YodB to the runoff transcription reaction mixtures showed that YodB could repress transcription from P₃ in vitro (Fig. 4) but not when template DNA was made from T–26A, T–20G, or T–19G DNA. The A–14T template showed reduced YodB-dependent repression compared to +40 deletion P₃ DNA. YodB could repress transcription of a template made from T24C mutant DNA or from DNA of the deletion mutant +5 (lacking DNA 3' from position +5). In contrast, PerR could repress the transcription of DNA made from the +40 deletion, T–26A, T–20G, T–19G, or A–14T spx P₃ DNA but not from +5 or from T24C. Neither repressor significantly affected transcription from the control rpsD (ribosomal protein S4) promoter DNA. These results suggested that sequences in the spx P₃ promoter were required for YodB-dependent repression, whereas sequences 3' of the spx P₃ start site were necessary for PerR-dependent repression. As predicted from work described in the accompanying paper, the in vitro transcription analysis uncovered two cis-acting elements, one being the site of the YodB interaction and one required for PerR-dependent control (15a).

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FIG. 4. Runoff in vitro transcription analysis showed the effects of purified PerR and YodB on the levels of spx transcripts. Linear DNA templates for spx (deletions at positions +40 and +5 and point mutations T24C, T–26A, T–20G, T–19G, and A–14T) promoters and rpsD were generated by PCR. RNAP and DNA templates were incubated with or without PerR or YodB as indicated. The major transcript (bottom band) was quantified by using IMAGEQUANT data analysis software. The ratio of transcription (percent) was measured by comparing the transcripts from the reaction without repressor protein (as 100%) to transcripts with repressor protein for each template.

Further verification of the roles of YodB and PerR in the control of spx transcription initiation and the respective cis-acting elements was obtained by examining the in vivo basal level of spx expression using the spx-bgaB fusion construct. Figure 5 shows the expression of mutant spx-bgaB fusions bearing mutations in the regulatory cis-acting sites in wild-type cells and perR (Fig. 5A) and yodB (Fig. 5B) mutant cells. Only the two mutations downstream of the transcription start site significantly lowered the ratio of basal bgaB activity in perR versus wild-type cells (Fig. 5A), while the upstream mutations residing in the P₃ promoter affected the ratio of yodB versus wild-type expression. These data confirm that YodB acts on the upstream cis regulatory sequence, while PerR acts on the negative control element located 3' to the transcription start site.

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FIG. 5. Effect of the perR (A) and yodB (B) disruption on the expression of spx promoter P3 point mutation cells. Cells were grown in DSM. The expression was determined as BgaB activity in Miller units at 30 min after cultures reached mid-log phase. Cells containing wild-type spx and point mutation (T–26A, T–20G, T–19G, A–14T, A3G, T7C, and T24C) promoters are indicated by white bars. Expression of the perR disruption and the perR disruption in spx point mutation cells is indicated by black bars (A). Expression of the yodB disruption and the yodB disruption in spx point mutation cells is indicated by gray bars (B). Results are means ± standard deviations from at least three independent experiments.

YodB and PerR proteins interact with two distinct regions of spx P₃ promoter DNA. DNase I footprinting analysis was performed to confirm that the regions of spx P₃ DNA required for transcriptional repression in vitro were sites of YodB and PerR interactions. End-labeled P₃ promoter DNA was combined with YodB protein, PerR protein, or both, followed by digestion with DNase I, denaturing gel electrophoresis, and phosphorimaging. As predicted from mutational and in vitro transcriptional analyses, PerR interacted with DNA 3' to the start site of P₃ transcript synthesis, protecting an area from approximately positions –3 to +35 (Fig. 6A). YodB protected a region from positions –3 to –32 (Fig. 6A, lanes 8 to 10), which encompasses the nucleotide positions that are the sites of mutations that affect YodB-dependent repression.

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FIG. 6. Result of DNase I footprinting of PerR and YodB to the top strand of the spx promoter. The wild-type spx promoter (A), point mutation T–26A spx promoter (B), point mutation T–19G spx promoter (C), and point mutation T+24C (lanes 1 to 3) and the wild type (lanes 4 to 6) (D) prepared by PCR were incubated in separate reaction mixtures with an increased amount of His-tagged PerR and YodB and subjected to DNase I cleavage. Lines in A indicate the protected regions, with a single line for PerR and double lines for YodB. The positions relative to the transcriptional start site are shown on the left (A and D). The nucleotide substitution (T24C) is indicated by an asterisk (D). BSA, bovine serum albumin.

Reduced binding of YodB was observed in footprinting reactions that contained spx promoter DNA with either the T–26A (Fig. 6B) or the T–19G (Fig. 6C and see Fig. 8) mutations, both of which give rise to elevated spx transcription in vivo. Neither mutation affected the binding of PerR (Fig. 6B and C). The mutation T24C, residing within the envelope of PerR contact (see Fig. 8), caused an altered DNase digestion pattern (Fig. 6D, compare lanes 1 and 4) and reduced the protection afforded by the PerR interaction (Fig. 6D, lanes 2 and 3 and lanes 5 and 6). These findings are in keeping with the in vivo and in vitro phenotypes of the spx P₃ mutations and the yodB and perR insertion mutations.

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FIG. 8. Protection patterns from DNase I footprinting experiments. The nucleotide sequences of the spx promoter are shown. The regions protected by YodB (double solid lines) and by PerR (single solid line) are indicated. Putative –10 and –35 sequences are boxed. TSS, transcription start site. RBS, ribosome-binding site.

Diamide and hydrogen peroxide reduce binding of YodB and PerR proteins to spx P₃ promoter DNA. The spx gene is a member of the PerR regulon (12), and we reasoned that the binding of PerR to spx promoter DNA might be sensitive to toxic oxidants such as diamide and H₂O₂. Using the DNase I protection assay, we determined whether the binding of PerR and YodB to P₃ was reduced if diamide or H₂O₂ was added to the footprinting reactions. As shown in lanes 1 to 5 of Fig. 7A, PerR bound poorly to the downstream operator sequence when diamide was present. The same result was observed when diamide was added to a binding reaction mixture containing YodB and spx P₃ promoter DNA (Fig. 7A, lanes 6 to 11). The binding of both PerR and YodB in combination is also impaired by diamide (Fig. 7A, lane 12). Diamide did not affect the interaction of the repressor CymR (3, 5) with one of its targets, the yrrT operon promoter (Fig. 7B), nor did it affect the activity of DNase I (Fig. 7A, lanes 1 and 2). The addition of hydrogen peroxide to the DNase I footprinting reaction mixtures (Fig. 7C) also impaired the binding of PerR (lanes 3 to 6), YodB (lanes 7 to 10), and YodB plus PerR (lanes 11 and 12) to the spx P3 promoter DNA. The concentrations used are slightly higher than the range of concentrations used in previous studies (10 mM to 75 mM) to inactivate the PerR repressor in vitro (13). Again, DNase I activity was not impaired by H₂O₂ at the higher concentration tested in the DNA-protein-binding experiments (Fig. 7C, lanes 1 and 2).

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FIG. 7. Effect of diamide and hydrogen peroxide on DNA binding. (A) DNase I footprinting was used to assess the effect of diamide on YodB and PerR binding to the spx promoter (A) and on CymR binding to the yrrT promoter (B). The protected region by CymR is indicated by the dashed line on the right side. The positions relative to the transcriptional start site are shown. (C) Effect of H2O2 on DNA binding. PerR and YodB were incubated with DNA as follows: lanes 1 and 2, DNA-alone control; lanes 3 to 6, 11, and 12, 1 µM PerR; lanes 7 to 12, 2 µM YodB. The concentrations of H2O2 used were 25 mM, 50 mM, and 100 mM. BSA, bovine serum albumin.

The above-described results suggest that hydrogen peroxide treatment would lead to an impairment of YodB/PerR-dependent negative control. Indeed, we observed increased spx P₃-directed ß-galactosidase activity in spx-lacZ fusion-bearing cells after treatment with 100 µM H₂O₂. Untreated cells showed an activity of 68.4 ± 1.8 Miller units and treated cells had activity of 98.7 ± 0.8 Miller units after 30 min H₂O₂ treatment.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The yjbC-spx operon is under complex transcriptional control involving the activities of four RNAP holoenzyme forms utilizing promoters in the yjbC promoter regions and in the intergenic region of yjbC and spx. Under the growth conditions used in the study reported here and in the accompanying paper (15a), transcription of spx was observed to be initiated from an intergenic promoter, P₃, that is recognized by the major ^A form of RNAP. Although a promoter recognized by ^M resides in the intergenic region, transcription from its start site was not detected in our experiments. The only promoter that was utilized in response to oxidative stress in our experiments was the P₃ promoter.

Studies described in the accompanying paper uncovered the P₃ promoter and two putative cis-acting negative control elements, mutations of which caused a derepression of spx transcription in vivo (15a). As shown herein, the two cis-acting sites are operators for two repressor proteins, PerR and YodB. The perR and yodB null mutations also lead to the derepression of spx transcription from P₃, and the products of the two genes interact directly with spx promoter DNA, as illustrated in Fig. 8. No cooperative interaction between YodB and PerR was detected by DNase I footprinting analysis (M. Leelakriangsak and P. Zuber, unpublished data). The binding of PerR and YodB to their operators can be reversed by the introduction of an oxidant, either hydrogen peroxide or diamide, to protein-DNA-binding reactions. We propose that spx is regulated at the level of gene transcription by the two repressors, which are inactivated by toxic oxidants.

Previous studies involving genome-wide analyses of gene expression have uncovered spx as a stress-induced gene. Proteomic and transcriptomic studies have shown that spx transcript levels increase in response to ethanol stress and phosphate limitation (1, 35). Mutations of yjbC and spx have been reported to confer a salt-stress-sensitive phenotype (30), although our attempts at repeating this result have not been successful. Transcriptomic analyses of the oxidative stress response have not uncovered the spx gene as being induced by reactive oxygen species, but the microarray analysis of the PerR regulon showed that spx transcript levels increased in a perR mutant (12). Additionally, treatment of cells with the thiol-specific oxidant diamide was previously reported to increase spx transcript levels (16). The accompanying paper provides data showing that diamide induction involves accelerated transcription from the P₃ promoter of spx (15a). This is likely due in part to the inactivation of PerR, which interacts with its operator located downstream of the P₃ transcriptional start site.

PerR possesses a zinc binding domain in which a zinc atom is coordinated by four cysteines in a CxxC...CxxC arrangement. The cysteines are quite resistant to oxidation due to the interaction with zinc, and only high concentrations of hydrogen peroxide can oxidize them in vitro with the release of zinc (14). No oxidation of the cysteines was observed in vivo (14), indicating that the major oxidant-sensing mechanism of PerR involves the histidines that coordinate ferrous ion (15). PerR is resistant to oxidation by diamide and releases zinc only when treated with the oxidant under harsh conditions (elevated temperature or in the presence of a denaturant) (14). Thus, the zinc binding domain is believed to serve a structural function rather than being a major oxidant-sensing domain of PerR. However, diamide treatment results in a loss of PerR binding to its operator in the spx P₃ promoter, which would implicate the zinc-coordinating cysteine residues as a redox target of the oxidant. Perhaps partial oxidation of the cysteines reduces the affinity of PerR for the operator in spx P₃.

The PerR protein used in the experiments reported here was obtained by purification under aerobic conditions that resulted in the loss of the Fe²⁺ cofactor by oxidation. This cofactor is required for the optimal reactivity of the His residue with peroxide (13, 15). The PerR protein of our studies is mostly devoid of Fe, as determined by inductively coupled plasma optical emission spectrometry (2 to 3% of total PerR protein is of the holo form) (data not shown). PerR is responsive to the presence of H₂O₂ in vitro (Fig. 7) but likely does not possess optimal reactivity.

YodB is a member of the DUF24 family of winged-helix transcription factors, of which B. subtilis HxlR (38) is the only member with a known regulatory function. HxlR activates the hxlAB operon, encoding the ribulose monophosphate pathway of formaldehyde fixation. HxlR is activated when cells encounter formaldehyde, but the mechanism of activation is not known. YodB is also similar to members of the ArsR family of transcriptional repressors, but it lacks the conserved cysteine residues in the H-T-H motif that function in metalloid coordination via trigonal geometry (10). Most close orthologs of YodB are found in bacilli, Listeria, Staphylococcus, and Clostridia. These orthologs, along with HxlR, have a conserved cysteine residue at or near position 6 at the N termini, but YodB is unique among them due to the presence of two cysteine residues near its C terminus. Like PerR, the addition of diamide or hydrogen peroxide to DNase I footprinting reactions resulted in a loss of DNA-binding activity. Current efforts are focused on the possible role of the cysteine residues in the redox control of YodB activity. The yodB gene resides adjacent to yodC, which encodes a putative nitroreductase. yodC and yodB are in divergent orientations with respect to one another, and preliminary analysis of yodC-lacZ expression has suggested that YodB is a repressor of yodC transcription (Leelakriangsak and Zuber, unpublished). The expression of the yodB and yodC genes is induced by catechol treatment (34), conditions that also induce the spx regulon. Paralogs of YodC include NfrA, an NAD(P)H-linked flavin binding nitroreductase that is encoded by a gene controlled by Spx and induced by heat shock and oxidative stress (19, 33). YodB might serve as another regulator that participates in the oxidative stress response of B. subtilis through its control of yodC and spx.

A curious observation is the reduction of spx transcript in the perR yodB double mutant when cells are treated with diamide. The total pleiotropic effects of the two mutations combined are unknown at this time, as are their effects on transcription of the yjbC-spx operon, apart from P₃. These questions are the subject of current investigations.

The study described herein has uncovered yet another level of control that governs the expression of spx. Previous reports detailed the redox control of Spx activity mediated by the N-terminal CxxC disulfide motif (24) and posttranscriptional control of Spx protein levels that includes proteolytic turnover of Spx by the ATP-dependent protease ClpXP (25, 27). The transcriptional control targeting the P₃ promoter represents another layer of regulation involving dual control by PerR and a previously unknown negative control factor, YodB. The conservation of Spx, PerR, and YodB among low-GC-content gram-positive species suggests an important network of control governing the bacterium's response to toxic oxidants.

 
We thank John D. Helmann and Mitsuo Ogura for gifts of strains and Michiko M. Nakano for helpful discussions and critical reading of the manuscript. We also thank Amanda Barry and Ninian Blackburn for performing inductively coupled plasma optical emission spectrometry on the PerR protein.

Research reported herein was supported by grant GM45898 from the National Institutes of Health and by a grant from the Medical Research Foundation of Oregon.

 
* Corresponding author. Mailing address: EBS/OGI School of Science and Engineering, Oregon Health and Science University, 20000 NW Walker Rd., Beaverton, OR 97229. Phone: (503) 748-7335. Fax: (503) 748-1464. E-mail: pzuber{at}ebs.ogi.edu.

Published ahead of print on 8 December 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, March 2007, p. 1736-1744, Vol. 189, No. 5
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