BACKGROUND
zinc is an essential trace element for all forms of life.
it serves as structural scaffold for protein folding and as cofactor for many enzymes and dna-binding proteins  <cit> .
however, due to its toxicity, mechanisms for zinc acquisition and efflux are tightly regulated according to metal ion requirements  <cit> .
in the gram positive bacterium bacillus subtilis, transcription of genes involved in zinc homeostasis is regulated by zur, a metalloprotein that binds zn as corepressor  <cit> .
in vivo, zur forms a homodimer that binds to a conserved dna motif, the zur box, which overlaps the σa-type promoter elements in target genes  <cit> .
dna-binding studies demonstrated that zur requires a minimal 9-1- <dig> inverted repeat motif for high-affinity binding  <cit> .
a stepwise activation model predicts that zur may respond to a wide range of intracellular zn concentrations to gradually repress the zur regulon  <cit> .
zur represses expression of genes encoding a high-affinity zinc abc transporter znuacb  <cit> , a putative low affinity zinc uptake system ycibc  <cit> , a gtp cyclohydrolase ib involved in folate biosynthesis foleb  <cit> , and zinc-independent alternative ribosomal proteins   <cit> .
in b. subtilis, zinc is also imported by the p-type atpase zosa, whose expression is controlled by the peroxide-sensing regulator, perr  <cit> .
induction of zosa in response to hydrogen peroxide stress leads to zn uptake, which plays an important protective role against oxidative stress damage  <cit> .
both zosa and znuacb zinc transporters are involved in the competence developmental process  <cit> .
zinc homeostasis is also maintained in b. subtilis thanks to a zinc-inducible efflux pump czcd important for growth in the presence of high concentration of zn  <cit> .
expression of this system is regulated at the transcriptional level by the metalloregulator czra  <cit> .
despite knowledge of zur-mediated regulation of zinc homeostasis, a global identification of the genes directly under zur control is still missing.
here, we used chromatin immunoprecipitation of zur-dna complexes coupled with hybridization of dna to tiled oligonucleotides arrays  to identify regions enriched for zur dna-binding sites in vivo, at the genomic scale.
we provide evidence that zur binds to  <dig> regions on the chromosome, including previously known promoter regions of the zur primary regulon as well as a number of inter- and intragenic regions.
half of the newly identified binding sites is associated with a predicted zur box.
we showed that an intragenic zur box was functional to mediate a zur-dependent repression when inserted in a promoter region.
in addition, several intragenic zur boxes were able to act as transcriptional cis-repressive element but the direct role of zur at intragenic sites remains unclear.
finally, our study suggests that zur binding to newly identified targets could be primed to fine-tune gene expression in interplay with other transcription factors in response to specific conditions such as the disulfide stress.
RESULTS
c-terminally spa-tagged zur is a functional regulator
the b. subtilis chromosome was modified at the zur locus to express zur fused at its c-terminus with the spa tag .
in the resulting zur::zur-spa strain, the zurspa protein is under the control of its native expression signals.
to examine the activity of the zurspa fusion protein, the ycic promoter region was fused with the lacz reporter gene and introduced at the amye locus in wild-type, zur::zur-spa and Δzur::apha <dig> strains  .
expression of ycic, monitored by measuring the β-galactosidase activities, was repressed in wild-type and zur::zur-spa cells  whereas it was increased by a 160-fold factor in Δzur cells.
thus, zurspa functionally repressed ycic expression as wild-type zur.table  <dig>
b. subtilis
strains used in this study
strain
genotype
a
source
trp
+
zur::zur-spa erm
amye::pycic’-lacz cat
amye::pycic’-lacz cat zur-spa erm
amye::pycic’-lacz cat Δzur::apha3
amye::paymad’-lacz cat
amye::pbymad’-lacz cat
amye::paydeo’-lacz cat
amye::pbydeo’-lacz cat
amye::paywhc’-lacz cat
amye::pbywhc’-lacz cat
amye::paktrd’-lacz cat
amye::pbktrd’-lacz cat
amye::payrpe’-lacz cat
amye::payrpe’-lacz cat Δzur::apha3
amye::pbyrpe’-lacz cat
amye::pbyrpe’-lacz cat Δzur::apha3
amye::paymad'-luc cat
amye::paymad'-luc cat Δspx::spc
amye::paymad'-luc cat Δzur::apha3
amye::pbymad'-luc cat
amye::pbymad'-luc cat Δzur::apha3
amye::pbymad’-lacz cat Δspx::spc
a
cat, pc <dig> chloramphenicol acetyl-transferase gene; apha <dig>  enterococcus faecalis kanamycin-resistance gene; erm, erythromycin-resistance gene; spc, staphylococcus aureus spectinomycin-resistance gene.
we further tested the effect of ion starvation on ycic'-lacz expression in the same genetic backgrounds.
the strains were cultivated in ms medium and samples were spread onto solid medium containing x-gal.
a drop of 100 μm edta, an ion chelating agent, was deposited at the center of the plates.
after incubation, a characteristic blue ring was observed around the edta drop  indicating that ion deficiency induced ycic expression in cells synthesizing zur as well as zurspa proteins.
the binding of zurspa to the ycic promoter region was reversible upon metal ion starvation.
we concluded from this data that the zurspa fusion protein was functional for transcriptional regulation.
genome-wide mapping of zur binding sites
to identify zur-binding targets in the b. subtilis genome, we carried out chip-on-chip experiments.
the zur::zur-spa strain was grown in lb medium to exponential phase.
after cross-linking, zur-bound dna was immunoprecipitated using a spa tag specific antibody.
the signals from two independent experiments were processed and peaks corresponding to zurspa binding sites were selected using a cut-off value of  <dig>  .
overall  <dig> enriched dna regions were identified from the chip-on-chip signals .
among them,  <dig> were located in genomic regions known to belong to the zur regulon   <cit> .
as in parallel, we used the chip-on-chip methodology to study various b. subtilis transcription factors  <cit> , we observed that all the peaks detected with zur were exclusively specific to this regulator.figure  <dig>
typical zur-binding sites during growth of
b. subtilis
in exponential phase in lb medium.
each of the  <dig> panels represents a chromosomal region with annotated genes.
the chip-on-chip profiles with the peaks detected are represented in blue.
zurspa-binding sites in promoter regions of the known zur regulon.
zur-binding sites in intragenic regions.
we further compared our data with previous in silico studies  <cit> .
the promoter-proximal zur-binding sites upstream of yrpe and the pseudogene s <dig>  exhibited zur box motifs.
we then tested expression of the yrpe gene in wild-type and Δzur cells by using a transcriptional fusion between the yrpe promoter region and the lacz reporter gene.
expression of the pyrpe promoter was 30-fold increased in a zur mutant compared to the wild-type during the exponential phase of growth .
these results demonstrated the zur-dependent regulation of yrpe.
we also detected zur binding upstream of the short yczl gene, encoding a protein of unknown function.
expression of yczl is co-regulated with ycic and is driven from a single promoter leading to the yczlycic transcript  <cit> .
altogether, these findings delineated the zur primary regulon, which is now composed of  <dig> genes expressed from  <dig> distinct promoters  fulfilling three criteria:  in vivo zur binding in chip-on-chip experiments;  presence of a zur box;  zur-dependent regulation of expression.
in addition,  <dig> additional promoter-proximal zur binding sites were detected less than  <dig> base-pairs upstream of a translational start site  suggesting a zur-dependent expression and existence of new candidates to be part of the zur regulon.
the expression of all the genes belonging to the zur primary regulon displayed a similar expression profile whereas the expression of the genes closed to the  <dig> newly identified promoter-proximal sites did not appear correlated with the zur primary regulon  <cit> .
these genes might be controlled by zur under specific unknown conditions.
the presence of predicted zur boxes in these regions is discussed below.
finally,  <dig> peaks were located within intragenic regions more than 200 bp downstream a start codon .
the location of these sites was intriguing since none zur intragenic binding site has so far been described.
prediction of zur boxes within zur-binding sites
to investigate the presence of zur boxes within the newly identified zur-binding sites, we performed in silico analyses.
zur binds to dna sites, which display high sequence similarity to those recognized by fur and perr although regulon overlap in naturally occurring promoter/operator sites has not been reported  <cit> .
here, we used the meme standard bioinformatic method  <cit>  to identify common motifs among genomic regions representing 100 bp centered at each zur-binding sites.
an appealing feature of this program is its ability to automatically compute optimal motif widths, in contrast to the majority of current motif-finding software.
we did not impose a constraint that the motif must be an inverted repeat sequence on the search.
this yielded a 20-nt motif present in  <dig> zur-binding sites and matching the previously reported zur box consensus   <cit> .
the conserved nucleotides at positions  <dig>   <dig>   <dig> and  <dig> corresponded to the most critical for zur binding   <cit> .figure  <dig>
zur box motifs present in zur-binding sites identified in chip-on-chip experiments.
the 20 nt palindromic consensus sequences identified from the in vivo zur binding sites.
the size of the nucleotide at each position indicates its relative prevalence in sequences used as training set for meme algorithm  <cit> .
alignment of the motif identified in this work with the previously reported zur box, fur box and perr box.
common nucleotides are indicated in bold.
alignment of zur box sequences:  zur boxes in promoter regions of genes belonging to the zur regulon;  intragenic zur boxes in zur binding regions detected by chip-on-chip.
nucleotides matching the consensus  and nucleotides different from the consensus  are indicated.
stars mark the critical positions for zur binding as previously described  <cit> .
seven predicted zur boxes occurred in the promoter regions belonging to the zur regulon, validating our in silico approach.
the  <dig> newly identified zur boxes were associated to in vivo zur binding in inter- as well as in intragenic regions with a broad distribution of chipscores , which reflect the strength of in vivo zur-dna interaction .
correlation between chipscores and the degree of similarity of each zur box to the consensus sequence was investigated.
as shown in figure  <dig>  no clear correlation was observed.
it is possible that differences in chipscores result not only from zur-dna binding affinity but also from differences in cross-linking efficiency at some dna regions.
interestingly, the highest chipscores  were associated to zur boxes located less than 50 bp from a translational start site  suggesting that the genomic location could contribute to the strength of zur binding in vivo.figure  <dig>
relationship between zur binding
in vivo
and similarity to the zur box consensus.
the chipscore is scored against the number of nucleotides matching the 20 nt-consensus sequence.
zur box motifs located in intragenic regions  and zur box motifs located in intergenic regions  are indicated.
stars indicate the zur box motifs belonging to the zur regulon as previously characterized  <cit> .
relationship between zur binding
in vivo
and the position of the detected zur-binding sites.
the chipscore is plotted against the position of the zur binding sites on the chromosome relative to the closest translational start sites.
black circles, zur-binding sites in a region containing a predicted zur box motif; white squares, zur-binding sites without significant match to the zur box consensus.
next, the zur box consensus was submitted to fimo  <cit>  to identify putative zur box in all the b. subtilis genome.
hits with a p value of ≤ 10− <dig> were regarded as significant, resulting in  <dig> candidates including the  <dig> zur boxes associated with in vivo zur binding  .
these results showed that only a set of potential zur boxes were bound by zur, at least in the conditions used.
half of the zur-binding sites detected by chip-on-chip did not display a significant match to the zur box consensus.
as shown in figure  <dig>  these sites were located in inter- as well as in intragenic with some high chipscores .
using meme, we were unable to identify a common dna sequence motif among zur targets that lack a canonical zur box motif.
the intragenic zur box from ymad promotes a zur-mediated repression when placed within a promoter region
we further investigated the functionality of an intragenic zur box to be recognized by the wild-type zur protein to promote a repressive effect on transcription.
we chose the intragenic zur box located in the ymad gene encoding a putative peroxiredoxin.
peroxiredoxins are important for antioxidant defense by reducing hydrogen peroxide , which induces the zinc uptake system zosa in b. subtilis  <cit> .
a Δymad deleted strain  was constructed and the sensitivity of this ymad mutant to h2o <dig> was tested in liquid medium.
after 30 min of growth in the presence of 400 μm h2o <dig>   <dig> % and 1% survival was observed for the mutant and the wild-type cells, respectively .
thus, deletion of the ymad gene increased the sensitivity of the cells to h2o <dig>  indicating that ymad plays a role in protecting cells against oxidative stress.
subsequently, we tested the ability of the ymad intragenic zur box to promote zur-mediated regulation.
in a previous in silico prediction, a unique zur box motif was detected in the yrpe promoter region from nucleotide − <dig> to − <dig> relative to the translational start site  <cit> .
yrpe is transcribed from a σa-dependent promoter   <cit> .
two types of transcriptional fusions with the promoter region of the yrpe gene were constructed.
first a payrpe'-lacz fusion was obtained using the yrpe promoter sequence spanning from − <dig> to − <dig> related to the translational start site, which included the native zur box sequence.
the fusion sequence was integrated at the amye locus of the wild-type and Δzur strains .
expression of the payrpe promoter was 20-fold increased in a Δzur mutant compared to the wild-type .
in the second pbyrpe'-lacz fusion, the native zur box of yrpe was replaced by the intragenic zur box sequence from ymad .
this fusion exhibited 16-fold higher level in Δzur cells than in the wild-type .
thus, the intragenic zur box sequence from ymad was recognized by zur to mediate transcriptional repression when located in a promoter region.
this confirmed the hypothesis that intragenic zur box sequences are functional zur binding sites.figure  <dig>
structure of the
yrpe
promoter region and expression of
yrpe
’-
lacz
transcriptional fusions.
detailed view of the yrpe promoter region in the two transcriptional fusions.
the σa-dependent recognition site is underlined, the ribosome-binding site is capitalized.
the zur box motif is bold.
in payrpe, the zur box motif is the native sequence.
in pbyrpe, the zur box motif is the intragenic zur box sequence from ymad.
the six nucleotides differences are indicated in bold capital letters.
expression of the transcriptional payrpe’-lacz and pbyrpe’-lacz fusions in the wild-type  strain and the Δzur deleted mutant.
nucleotides are numbered relative to the translational start site of the yrpe gene.
location of the zur motif is indicated by boxes.
white box, native zur box sequence from the yrpe promoter region; black box, zur box sequence identified within the ymad gene.
β-galactosidase activities were measured at least  <dig> times independently.
intragenic zur boxes: a role in transcription elongation?
some transcription factors, such as b. subtilis cody and ccpa regulators, can regulate the transcription elongation by a roadblock mechanism  <cit> .
thus, we tested whether several intragenic zur binding sites could participate in such a regulatory process.
the ydeo, ywhc and ktrd genes contain intragenic zur-binding sites and encode putative membrane protein, potential zinc metalloprotease, and k+-transporting atpase, respectively.
they are transcribed from σa-dependent promoters  <cit> .
different dna regions were fused to the lacz reporter gene and integrated at the amye locus of the wild-type strain .
the paymad, paydeo, paywhc and paktrd fusions contained the promoter region and the start of the encoding-sequence including the native zur box.
the pbymad, pbydeo, pbywhc and pbktrd fusions contained symmetric mutations in each half-site of the zur box at position  <dig> and  <dig>  because these point- mutations were shown to completely abrogate zur binding .
β-galactosidase activities were examined during exponential growth .
expression values of the paymad, paydeo, paywhc and paktrd fusions ranged between  <dig> and  <dig> units of β-galactosidase activity.
in contrast, the pbymad, pbydeo, pbywhc and pbktrd fusions were 4- to 9-fold upregulated  suggesting that intragenic zur boxes mediate a transcriptional cis-repressive effect.
expression of the pa and pb fusions was also tested in a zur mutant but similar levels of expression was observed in Δzur and wild-type cells .figure  <dig>
expression of various transcriptional fusions in the wild-type and the Δ
zur
strains.
scheme of the dna regions inserted upstream from the lacz reporter gene.
nucleotides are numbered relative to the translational start site of the ymad, ywhc, ydeo and ktrd genes.
the native intragenic zur boxes are indicated by black boxes.
the mutated intragenic zur boxes are indicated by white boxes.
the generated point-mutations in the zur box sequences are indicated in bold capital letters.
cells were grown in lb medium.
β-galactosidase activities were determined in extracts prepared from exponentially growing cells at od <dig> of  <dig> .
zur-binding sites overlap genes responding to disulfide stress
our analyses revealed that a large overlap existed between the location of zur-binding sites and genes whose expression responds to disulfide stress  <cit> .
zur-binding sites were located less than 400 bp upstream of the translational start site or in the coding sequences of 31-activated and 19-repressed genes in response to diamide stress .
furthermore, zur-binding sites are associated to  <dig> genes  reported to be directly regulated by spx  <cit> , suggesting that zur- and spx-mediated regulations partially overlap.
to investigate a potential role of an intragenic zur box to mediate a disulphide stress response, we choose to test the effect of diamide treatment on ymad expression in wild-type and Δspx genetic backgrounds.
as it is known that lacz activity is very sensitive to diamide addition, we constructed a paymad'-luc fusion between the region from nucleotide − <dig> to + <dig> relative to ymad translational start site and the luciferase gene.
a second pbymad'-luc fusion was constructed containing point-mutations in the intragenic zur box.
luciferase activity was recorded during the growth in lb medium.
expression of pbymad'-luc was 10-fold higher than expression of paymad'-luc  confirming the results observed with the lacz fusions .figure  <dig>
expression of
ymad
in various genetic backgrounds and after diamide treatment.
expression of paymad and pbymad fusions in the wild-type  strain , in Δzur  or Δspx cells .
growth was monitored by measuring the optical density at 600 nm: grey line, wild-type.
the Δzur and Δspx stains grew with the same growth profile than the wild-type.
promoter activity of paymad and pbymad without diamide treatment , with  <dig>  mm diamide  or with  <dig>  mm diamide .
final concentration of  <dig>  or  <dig>  mm diamide was added at t = 1 hour when cells reached an od <dig> of  <dig>  in lb medium.
for each strain, one representative curve, out of three independent replicates realized, is shown.
no expression of paymad'-luc and pbymad'-luc was detected in a Δspx mutant , in keeping with the spx-dependent activation of ymad  <cit> .
in the wild-type cells,  <dig>  or  <dig>  mm diamide treatment reproducibly increased paymad'-luc expression .
this indicated that zur binding to the zur box of paymad did not interfere woth spx activity.
the same pattern of induction was observed with pbymad .
thus, the intragenic zur box did not appear involved in the spx-dependent upregulation.
discussion
using the chip-on-chip methodology, we identified  <dig> enriched dna-regions in the b. subtilis chromosome that are reproducibly bound by the zur repressor under abundant zinc growth conditions.
we recovered the known zur regulon and confirmed that the predicted zur boxes present upstream of yrpe and rpmgc  <cit>  are functional binding sites in vivo.
these data allow to define the zur primary regulon, which is now composed of  <dig> transcription units.
consequently, the whole zur binding sites identified by chip-on-chip appear relevant.
remarkably, a second set of  <dig> newly identified sites bound by zur contained a zur box motif, which differs from the fur and perr boxes at positions  <dig>   <dig>   <dig>  and  <dig> and displays conservation of bases at flanking positions that are not strongly conserved in fur and perr boxes    <cit> .
the last set of zur binding dna sites did not contain any direct-repeat sequence or any common motif by the standard bioinformatic methods that we used, suggesting that zur recognizes degenerated zur box sequences or that other factors are required for zur binding at these sites.
interestingly, the zur binding sites with and without predicted zur box exhibit a similar distribution relative to coding sequences, with an enrichment around the translation start site , suggesting that both types of sites are functionally similar.
a surprising result from this study was the finding that  <dig> zur binding sites are located in intragenic regions.
sixteen of those contain a predicted zur box.
this contrasts sharply with in silico studies, which tend to consider transcription factor-targets within coding sequences as artefacts.
analysis with transcriptional fusions using the lacz reporter gene allowed us to show that the intragenic zur box sequence from the ymad gene was fully functional for zur-mediated repression when placed in a promoter region.
thus, the experimental strategies used in this study revealed unexpected and functional zur binding sites.
in addition, we showed that the intragenic zur box within ymad, ydeo, ywhc and ktrd genes had a cis-repressive effect  on transcription.
expression of these genes was also tested in a Δzur mutant but similar levels of expression was observed in Δzur and wild-type cells .
as disruption of zinc homeostasis in Δzur cells likely affects many cellular processes, the direct role of zur binding to the intragenic zur boxes could be masked by other regulatory effects.
our study highlights the presence of zur boxes within a subset of genes encoding functions related to metal ion homeostasis or oxidative stress.
we showed that the ymad gene codes for putative peroxiredoxin-related protein, which plays a role in protecting cells against oxidative stress.
interestingly, ymad is also under direct regulation by spx  <cit>  a global transcription key regulator for maintaining redox homeostasis of b. subtilis cells exposed to disulfide stress  <cit> .
the intragenic zur box of ymad did not appear to play a direct role on the spx-dependent regulation.
remarkably, we observed that the ymad transient induction upon exposure to diamide was abolished in Δzur cells  pointing to an interconnection of the zur- and spx-mediated responses.
as the degradation of spx is ensured by the clpxp protease  <cit> , disruption of zinc homeostasis in Δzur cells could impact on the activity of the zn-dependent protein clpx and, as a consequence, on the turnover of spx.
overall, it was not possible to conclude about the direct role of zur to mediate a regulation via the intragenic zur boxes because of Δzur deletion may entail significantly changes in other transcription factors activity.
in this intricate regulatory network, the binding of zur to intragenic zur boxes may contribute to fine-tune gene expression in response to zinc availability.
the interconnection between zur and spx may involve more than one gene as zur binding sites were detected within or near  <dig> spx-regulated genes and overall near  <dig> genes responsive to diamide stress .
overlap between disulfide and oxidative stress responses was previously identified in b. subtilis for the kata gene encoding a catalase.
expression of kata is under the dual control of perr  <cit>  and spx  <cit>  regulators.
in addition, a regulatory interplay between the responses to zinc deprivation and disulfide stress has been described in streptomyces coelicolor, where the activity of the thiol-disulfide metabolism regulator σr is induced upon zinc limitation  <cit> .
our results emphasize the complex interplay between the regulatory networks controlling zinc homeostasis and redox homeostasis, especially the oxidative and disulfide stress responses.
CONCLUSIONS
the chip-on-chip approach used in this study allowed to considerably expand the catalogue of in vivo zur-binding sites to  <dig> inter- as well as intragenic regions.
half of those is associated with an in silico predicted zur box.
the binding of zur to the newly identified targets may contribute to fine-tune gene expression under specific conditions, our results highlighting a complex link between zur and the disulfide stress response.
intragenic zur boxes could be involved in an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors.
future investigations will be required to investigate the role of zur binding sites in transcriptional regulation.

 zinc is an essential trace element for all forms of life. it serves as structural scaffold for protein folding and as cofactor for many enzymes and dna-binding proteins. however, due to its toxicity, mechanisms for zinc acquisition and efflux are tightly regulated according to metal ion requirements. in the gram positive bacterium bacillus subtilis, transcription of genes involved in zinc homeostasis is regulated by zur, a metalloprotein that binds zn as corepressor. in vivo, zur forms a homodimer that binds to a conserved dna motif, the zur box, which overlaps the σa-type promoter elements in target genes. dna-binding studies demonstrated that zur requires a minimal 9-1- inverted repeat motif for high-affinity binding. a stepwise activation model predicts that zur may respond to a wide range of intracellular zn concentrations to gradually repress the zur regulon. zur represses expression of genes encoding a high-affinity zinc abc transporter znuacb, a putative low affinity zinc uptake system ycibc, a gtp cyclohydrolase ib involved in folate biosynthesis foleb, and zinc-independent alternative ribosomal proteins. in b. subtilis, zinc is also imported by the p-type atpase zosa, whose expression is controlled by the peroxide-sensing regulator, perr. induction of zosa in response to hydrogen peroxide stress leads to zn uptake, which plays an important protective role against oxidative stress damage. both zosa and znuacb zinc transporters are involved in the competence developmental process. zinc homeostasis is also maintained in b. subtilis thanks to a zinc-inducible efflux pump czcd important for growth in the presence of high concentration of zn. expression of this system is regulated at the transcriptional level by the metalloregulator czra. despite knowledge of zur-mediated regulation of zinc homeostasis, a global identification of the genes directly under zur control is still missing. here, we used chromatin immunoprecipitation of zur-dna complexes coupled with hybridization of dna to tiled oligonucleotides arrays to identify regions enriched for zur dna-binding sites in vivo, at the genomic scale. we provide evidence that zur binds to regions on the chromosome, including previously known promoter regions of the zur primary regulon as well as a number of inter- and intragenic regions. half of the newly identified binding sites is associated with a predicted zur box. we showed that an intragenic zur box was functional to mediate a zur-dependent repression when inserted in a promoter region. in addition, several intragenic zur boxes were able to act as transcriptional cis-repressive element but the direct role of zur at intragenic sites remains unclear. finally, our study suggests that zur binding to newly identified targets could be primed to fine-tune gene expression in interplay with other transcription factors in response to specific conditions such as the disulfide stress.  c-terminally spa-tagged zur is a functional regulator the b. subtilis chromosome was modified at the zur locus to express zur fused at its c-terminus with the spa tag. in the resulting zur::zur-spa strain, the zurspa protein is under the control of its native expression signals. to examine the activity of the zurspa fusion protein, the ycic promoter region was fused with the lacz reporter gene and introduced at the amye locus in wild-type, zur::zur-spa and Δzur::apha strains. expression of ycic, monitored by measuring the β-galactosidase activities, was repressed in wild-type and zur::zur-spa cells whereas it was increased by a 160-fold factor in Δzur cells. thus, zurspa functionally repressed ycic expression as wild-type zur.table b. subtilis strains used in this study strain genotype a source trp + zur::zur-spa erm amye::pycic’-lacz cat amye::pycic’-lacz cat zur-spa erm amye::pycic’-lacz cat Δzur::apha3 amye::paymad’-lacz cat amye::pbymad’-lacz cat amye::paydeo’-lacz cat amye::pbydeo’-lacz cat amye::paywhc’-lacz cat amye::pbywhc’-lacz cat amye::paktrd’-lacz cat amye::pbktrd’-lacz cat amye::payrpe’-lacz cat amye::payrpe’-lacz cat Δzur::apha3 amye::pbyrpe’-lacz cat amye::pbyrpe’-lacz cat Δzur::apha3 amye::paymad'-luc cat amye::paymad'-luc cat Δspx::spc amye::paymad'-luc cat Δzur::apha3 amye::pbymad'-luc cat amye::pbymad'-luc cat Δzur::apha3 amye::pbymad’-lacz cat Δspx::spc a cat, pc chloramphenicol acetyl-transferase gene; apha enterococcus faecalis kanamycin-resistance gene; erm, erythromycin-resistance gene; spc, staphylococcus aureus spectinomycin-resistance gene. we further tested the effect of ion starvation on ycic'-lacz expression in the same genetic backgrounds. the strains were cultivated in ms medium and samples were spread onto solid medium containing x-gal. a drop of 100 μm edta, an ion chelating agent, was deposited at the center of the plates. after incubation, a characteristic blue ring was observed around the edta drop indicating that ion deficiency induced ycic expression in cells synthesizing zur as well as zurspa proteins. the binding of zurspa to the ycic promoter region was reversible upon metal ion starvation. we concluded from this data that the zurspa fusion protein was functional for transcriptional regulation. genome-wide mapping of zur binding sites to identify zur-binding targets in the b. subtilis genome, we carried out chip-on-chip experiments. the zur::zur-spa strain was grown in lb medium to exponential phase. after cross-linking, zur-bound dna was immunoprecipitated using a spa tag specific antibody. the signals from two independent experiments were processed and peaks corresponding to zurspa binding sites were selected using a cut-off value of. overall enriched dna regions were identified from the chip-on-chip signals. among them, were located in genomic regions known to belong to the zur regulon. as in parallel, we used the chip-on-chip methodology to study various b. subtilis transcription factors, we observed that all the peaks detected with zur were exclusively specific to this regulator.figure typical zur-binding sites during growth of b. subtilis in exponential phase in lb medium. each of the panels represents a chromosomal region with annotated genes. the chip-on-chip profiles with the peaks detected are represented in blue. zurspa-binding sites in promoter regions of the known zur regulon. zur-binding sites in intragenic regions. we further compared our data with previous in silico studies. the promoter-proximal zur-binding sites upstream of yrpe and the pseudogene s exhibited zur box motifs. we then tested expression of the yrpe gene in wild-type and Δzur cells by using a transcriptional fusion between the yrpe promoter region and the lacz reporter gene. expression of the pyrpe promoter was 30-fold increased in a zur mutant compared to the wild-type during the exponential phase of growth. these results demonstrated the zur-dependent regulation of yrpe. we also detected zur binding upstream of the short yczl gene, encoding a protein of unknown function. expression of yczl is co-regulated with ycic and is driven from a single promoter leading to the yczlycic transcript. altogether, these findings delineated the zur primary regulon, which is now composed of genes expressed from distinct promoters fulfilling three criteria: in vivo zur binding in chip-on-chip experiments; presence of a zur box; zur-dependent regulation of expression. in addition, additional promoter-proximal zur binding sites were detected less than base-pairs upstream of a translational start site suggesting a zur-dependent expression and existence of new candidates to be part of the zur regulon. the expression of all the genes belonging to the zur primary regulon displayed a similar expression profile whereas the expression of the genes closed to the newly identified promoter-proximal sites did not appear correlated with the zur primary regulon. these genes might be controlled by zur under specific unknown conditions. the presence of predicted zur boxes in these regions is discussed below. finally, peaks were located within intragenic regions more than 200 bp downstream a start codon. the location of these sites was intriguing since none zur intragenic binding site has so far been described. prediction of zur boxes within zur-binding sites to investigate the presence of zur boxes within the newly identified zur-binding sites, we performed in silico analyses. zur binds to dna sites, which display high sequence similarity to those recognized by fur and perr although regulon overlap in naturally occurring promoter/operator sites has not been reported. here, we used the meme standard bioinformatic method to identify common motifs among genomic regions representing 100 bp centered at each zur-binding sites. an appealing feature of this program is its ability to automatically compute optimal motif widths, in contrast to the majority of current motif-finding software. we did not impose a constraint that the motif must be an inverted repeat sequence on the search. this yielded a 20-nt motif present in zur-binding sites and matching the previously reported zur box consensus. the conserved nucleotides at positions and corresponded to the most critical for zur binding.figure zur box motifs present in zur-binding sites identified in chip-on-chip experiments. the 20 nt palindromic consensus sequences identified from the in vivo zur binding sites. the size of the nucleotide at each position indicates its relative prevalence in sequences used as training set for meme algorithm. alignment of the motif identified in this work with the previously reported zur box, fur box and perr box. common nucleotides are indicated in bold. alignment of zur box sequences: zur boxes in promoter regions of genes belonging to the zur regulon; intragenic zur boxes in zur binding regions detected by chip-on-chip. nucleotides matching the consensus and nucleotides different from the consensus are indicated. stars mark the critical positions for zur binding as previously described. seven predicted zur boxes occurred in the promoter regions belonging to the zur regulon, validating our in silico approach. the newly identified zur boxes were associated to in vivo zur binding in inter- as well as in intragenic regions with a broad distribution of chipscores, which reflect the strength of in vivo zur-dna interaction. correlation between chipscores and the degree of similarity of each zur box to the consensus sequence was investigated. as shown in figure  no clear correlation was observed. it is possible that differences in chipscores result not only from zur-dna binding affinity but also from differences in cross-linking efficiency at some dna regions. interestingly, the highest chipscores were associated to zur boxes located less than 50 bp from a translational start site suggesting that the genomic location could contribute to the strength of zur binding in vivo.figure relationship between zur binding in vivo and similarity to the zur box consensus. the chipscore is scored against the number of nucleotides matching the 20 nt-consensus sequence. zur box motifs located in intragenic regions and zur box motifs located in intergenic regions are indicated. stars indicate the zur box motifs belonging to the zur regulon as previously characterized. relationship between zur binding in vivo and the position of the detected zur-binding sites. the chipscore is plotted against the position of the zur binding sites on the chromosome relative to the closest translational start sites. black circles, zur-binding sites in a region containing a predicted zur box motif; white squares, zur-binding sites without significant match to the zur box consensus. next, the zur box consensus was submitted to fimo to identify putative zur box in all the b. subtilis genome. hits with a p value of ≤ 10− were regarded as significant, resulting in candidates including the zur boxes associated with in vivo zur binding. these results showed that only a set of potential zur boxes were bound by zur, at least in the conditions used. half of the zur-binding sites detected by chip-on-chip did not display a significant match to the zur box consensus. as shown in figure  these sites were located in inter- as well as in intragenic with some high chipscores. using meme, we were unable to identify a common dna sequence motif among zur targets that lack a canonical zur box motif. the intragenic zur box from ymad promotes a zur-mediated repression when placed within a promoter region we further investigated the functionality of an intragenic zur box to be recognized by the wild-type zur protein to promote a repressive effect on transcription. we chose the intragenic zur box located in the ymad gene encoding a putative peroxiredoxin. peroxiredoxins are important for antioxidant defense by reducing hydrogen peroxide, which induces the zinc uptake system zosa in b. subtilis. a Δymad deleted strain was constructed and the sensitivity of this ymad mutant to h2o was tested in liquid medium. after 30 min of growth in the presence of 400 μm h2o % and 1% survival was observed for the mutant and the wild-type cells, respectively. thus, deletion of the ymad gene increased the sensitivity of the cells to h2o indicating that ymad plays a role in protecting cells against oxidative stress. subsequently, we tested the ability of the ymad intragenic zur box to promote zur-mediated regulation. in a previous in silico prediction, a unique zur box motif was detected in the yrpe promoter region from nucleotide − to − relative to the translational start site. yrpe is transcribed from a σa-dependent promoter. two types of transcriptional fusions with the promoter region of the yrpe gene were constructed. first a payrpe'-lacz fusion was obtained using the yrpe promoter sequence spanning from − to − related to the translational start site, which included the native zur box sequence. the fusion sequence was integrated at the amye locus of the wild-type and Δzur strains. expression of the payrpe promoter was 20-fold increased in a Δzur mutant compared to the wild-type. in the second pbyrpe'-lacz fusion, the native zur box of yrpe was replaced by the intragenic zur box sequence from ymad. this fusion exhibited 16-fold higher level in Δzur cells than in the wild-type. thus, the intragenic zur box sequence from ymad was recognized by zur to mediate transcriptional repression when located in a promoter region. this confirmed the hypothesis that intragenic zur box sequences are functional zur binding sites.figure structure of the yrpe promoter region and expression of yrpe ’- lacz transcriptional fusions. detailed view of the yrpe promoter region in the two transcriptional fusions. the σa-dependent recognition site is underlined, the ribosome-binding site is capitalized. the zur box motif is bold. in payrpe, the zur box motif is the native sequence. in pbyrpe, the zur box motif is the intragenic zur box sequence from ymad. the six nucleotides differences are indicated in bold capital letters. expression of the transcriptional payrpe’-lacz and pbyrpe’-lacz fusions in the wild-type strain and the Δzur deleted mutant. nucleotides are numbered relative to the translational start site of the yrpe gene. location of the zur motif is indicated by boxes. white box, native zur box sequence from the yrpe promoter region; black box, zur box sequence identified within the ymad gene. β-galactosidase activities were measured at least times independently. intragenic zur boxes: a role in transcription elongation? some transcription factors, such as b. subtilis cody and ccpa regulators, can regulate the transcription elongation by a roadblock mechanism. thus, we tested whether several intragenic zur binding sites could participate in such a regulatory process. the ydeo, ywhc and ktrd genes contain intragenic zur-binding sites and encode putative membrane protein, potential zinc metalloprotease, and k+-transporting atpase, respectively. they are transcribed from σa-dependent promoters. different dna regions were fused to the lacz reporter gene and integrated at the amye locus of the wild-type strain. the paymad, paydeo, paywhc and paktrd fusions contained the promoter region and the start of the encoding-sequence including the native zur box. the pbymad, pbydeo, pbywhc and pbktrd fusions contained symmetric mutations in each half-site of the zur box at position and because these point- mutations were shown to completely abrogate zur binding. β-galactosidase activities were examined during exponential growth. expression values of the paymad, paydeo, paywhc and paktrd fusions ranged between and units of β-galactosidase activity. in contrast, the pbymad, pbydeo, pbywhc and pbktrd fusions were 4- to 9-fold upregulated suggesting that intragenic zur boxes mediate a transcriptional cis-repressive effect. expression of the pa and pb fusions was also tested in a zur mutant but similar levels of expression was observed in Δzur and wild-type cells.figure expression of various transcriptional fusions in the wild-type and the Δ zur strains. scheme of the dna regions inserted upstream from the lacz reporter gene. nucleotides are numbered relative to the translational start site of the ymad, ywhc, ydeo and ktrd genes. the native intragenic zur boxes are indicated by black boxes. the mutated intragenic zur boxes are indicated by white boxes. the generated point-mutations in the zur box sequences are indicated in bold capital letters. cells were grown in lb medium. β-galactosidase activities were determined in extracts prepared from exponentially growing cells at od of. zur-binding sites overlap genes responding to disulfide stress our analyses revealed that a large overlap existed between the location of zur-binding sites and genes whose expression responds to disulfide stress. zur-binding sites were located less than 400 bp upstream of the translational start site or in the coding sequences of 31-activated and 19-repressed genes in response to diamide stress. furthermore, zur-binding sites are associated to genes reported to be directly regulated by spx, suggesting that zur- and spx-mediated regulations partially overlap. to investigate a potential role of an intragenic zur box to mediate a disulphide stress response, we choose to test the effect of diamide treatment on ymad expression in wild-type and Δspx genetic backgrounds. as it is known that lacz activity is very sensitive to diamide addition, we constructed a paymad'-luc fusion between the region from nucleotide − to + relative to ymad translational start site and the luciferase gene. a second pbymad'-luc fusion was constructed containing point-mutations in the intragenic zur box. luciferase activity was recorded during the growth in lb medium. expression of pbymad'-luc was 10-fold higher than expression of paymad'-luc confirming the results observed with the lacz fusions.figure expression of ymad in various genetic backgrounds and after diamide treatment. expression of paymad and pbymad fusions in the wild-type strain, in Δzur or Δspx cells. growth was monitored by measuring the optical density at 600 nm: grey line, wild-type. the Δzur and Δspx stains grew with the same growth profile than the wild-type. promoter activity of paymad and pbymad without diamide treatment, with  mm diamide or with  mm diamide. final concentration of or  mm diamide was added at t = 1 hour when cells reached an od of in lb medium. for each strain, one representative curve, out of three independent replicates realized, is shown. no expression of paymad'-luc and pbymad'-luc was detected in a Δspx mutant, in keeping with the spx-dependent activation of ymad. in the wild-type cells, or  mm diamide treatment reproducibly increased paymad'-luc expression. this indicated that zur binding to the zur box of paymad did not interfere woth spx activity. the same pattern of induction was observed with pbymad. thus, the intragenic zur box did not appear involved in the spx-dependent upregulation. discussion using the chip-on-chip methodology, we identified enriched dna-regions in the b. subtilis chromosome that are reproducibly bound by the zur repressor under abundant zinc growth conditions. we recovered the known zur regulon and confirmed that the predicted zur boxes present upstream of yrpe and rpmgc are functional binding sites in vivo. these data allow to define the zur primary regulon, which is now composed of transcription units. consequently, the whole zur binding sites identified by chip-on-chip appear relevant. remarkably, a second set of newly identified sites bound by zur contained a zur box motif, which differs from the fur and perr boxes at positions and and displays conservation of bases at flanking positions that are not strongly conserved in fur and perr boxes. the last set of zur binding dna sites did not contain any direct-repeat sequence or any common motif by the standard bioinformatic methods that we used, suggesting that zur recognizes degenerated zur box sequences or that other factors are required for zur binding at these sites. interestingly, the zur binding sites with and without predicted zur box exhibit a similar distribution relative to coding sequences, with an enrichment around the translation start site, suggesting that both types of sites are functionally similar. a surprising result from this study was the finding that zur binding sites are located in intragenic regions. sixteen of those contain a predicted zur box. this contrasts sharply with in silico studies, which tend to consider transcription factor-targets within coding sequences as artefacts. analysis with transcriptional fusions using the lacz reporter gene allowed us to show that the intragenic zur box sequence from the ymad gene was fully functional for zur-mediated repression when placed in a promoter region. thus, the experimental strategies used in this study revealed unexpected and functional zur binding sites. in addition, we showed that the intragenic zur box within ymad, ydeo, ywhc and ktrd genes had a cis-repressive effect on transcription. expression of these genes was also tested in a Δzur mutant but similar levels of expression was observed in Δzur and wild-type cells. as disruption of zinc homeostasis in Δzur cells likely affects many cellular processes, the direct role of zur binding to the intragenic zur boxes could be masked by other regulatory effects. our study highlights the presence of zur boxes within a subset of genes encoding functions related to metal ion homeostasis or oxidative stress. we showed that the ymad gene codes for putative peroxiredoxin-related protein, which plays a role in protecting cells against oxidative stress. interestingly, ymad is also under direct regulation by spx a global transcription key regulator for maintaining redox homeostasis of b. subtilis cells exposed to disulfide stress. the intragenic zur box of ymad did not appear to play a direct role on the spx-dependent regulation. remarkably, we observed that the ymad transient induction upon exposure to diamide was abolished in Δzur cells pointing to an interconnection of the zur- and spx-mediated responses. as the degradation of spx is ensured by the clpxp protease, disruption of zinc homeostasis in Δzur cells could impact on the activity of the zn-dependent protein clpx and, as a consequence, on the turnover of spx. overall, it was not possible to conclude about the direct role of zur to mediate a regulation via the intragenic zur boxes because of Δzur deletion may entail significantly changes in other transcription factors activity. in this intricate regulatory network, the binding of zur to intragenic zur boxes may contribute to fine-tune gene expression in response to zinc availability. the interconnection between zur and spx may involve more than one gene as zur binding sites were detected within or near spx-regulated genes and overall near genes responsive to diamide stress. overlap between disulfide and oxidative stress responses was previously identified in b. subtilis for the kata gene encoding a catalase. expression of kata is under the dual control of perr and spx regulators. in addition, a regulatory interplay between the responses to zinc deprivation and disulfide stress has been described in streptomyces coelicolor, where the activity of the thiol-disulfide metabolism regulator σr is induced upon zinc limitation. our results emphasize the complex interplay between the regulatory networks controlling zinc homeostasis and redox homeostasis, especially the oxidative and disulfide stress responses.  the chip-on-chip approach used in this study allowed to considerably expand the catalogue of in vivo zur-binding sites to inter- as well as intragenic regions. half of those is associated with an in silico predicted zur box. the binding of zur to the newly identified targets may contribute to fine-tune gene expression under specific conditions, our results highlighting a complex link between zur and the disulfide stress response. intragenic zur boxes could be involved in an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. future investigations will be required to investigate the role of zur binding sites in transcriptional regulation. 
however, the disruption of zinc homeostasis in Δzur cells likely affects many cellular processes masking direct zur-dependent effects. subtilischip-on-chipzinc homeostasisdisulfide stressissue-copyright-statement© the author 2015 seven zur-binding regions constitute the zur primary regulon while newly identified targets were associated with a predicted zur box. in addition, intragenic zur boxes appeared to mediate a transcriptional cis-repressive effect but the function of zur at these sites remains unclear. zur binding to intragenic zur boxes could prime an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. the bacillus subtilis zur transcription factor recognizes a specific dna motif, the zur box, to repress expression of genes in response to zinc availability. using chromatin immunoprecipitation coupled with hybridization to dna tiling arrays, we reported the identification of inter- and intragenic chromosomal sites bound by zur. using transcriptional fusions an intragenic zur box was showed to promote a full zur-mediated repression when placed within a promoter region. our findings considerably expand the potential zur regulon, and reveal a new level of complexity in zur binding to its targets via a zur box motif and via a yet unknown mechanism that remains to be characterized. finally, most zur-binding sites were located near or within genes responsive to disulfide stress.
 the bacillus subtilis zur transcription factor recognizes a specific dna motif, the zur box, to repress expression of genes in response to zinc availability. although several zur-regulated genes are well characterized, a genome-wide mapping of zur-binding sites is needed to define further the set of genes directly regulated by this protein.  using chromatin immunoprecipitation coupled with hybridization to dna tiling arrays, we reported the identification of inter- and intragenic chromosomal sites bound by zur. seven zur-binding regions constitute the zur primary regulon while newly identified targets were associated with a predicted zur box. using transcriptional fusions an intragenic zur box was showed to promote a full zur-mediated repression when placed within a promoter region. in addition, intragenic zur boxes appeared to mediate a transcriptional cis-repressive effect but the function of zur at these sites remains unclear. zur binding to intragenic zur boxes could prime an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. however, the disruption of zinc homeostasis in Δzur cells likely affects many cellular processes masking direct zur-dependent effects. finally, most zur-binding sites were located near or within genes responsive to disulfide stress. these findings expand the potential zur regulon and reveal unknown interconnections between zinc and redox homeostasis regulatory networks.  our findings considerably expand the potential zur regulon, and reveal a new level of complexity in zur binding to its targets via a zur box motif and via a yet unknown mechanism that remains to be characterized. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords zur regulatorb. subtilischip-on-chipzinc homeostasisdisulfide stressissue-copyright-statement© the author 2015 