BACKGROUND
epidemiological trends indicate clinical acquisition of clostridium difficile as the primary route of human infection by this bacterium  <cit> .
the risk of c. difficile becoming a community-acquired infection is likely to increase without the development of better identification and more effective treatment  <cit> .
the genome of c. difficile has been described as “highly dynamic” based on the prevalence of horizontal gene transfer  <cit> .
the impact of a genome that readily changes in response to environmental stress could be a major indicator of c. difficile pathogenicity  <cit> .
c. difficile produces spores that allow it to be viable for extended periods, even in the presence of antibiotics, which could explain the persistence of this human pathogen during host-to-host transmission and in the hospital environment  <cit> .
transcription factors orchestrate the regulation of survival, proliferation, virulence, and antibiotic resistance mechanisms of human pathogens.
as part of our larger goal aimed at elucidating structure and function of transcription regulatory mechanisms involved in virulence and antibiotic resistance of human pathogens, we focused on protein targets from a hypervirulent strain of c. difficile .
herein, we present our results on a member of the padr family of transcription regulators  that we have named cdpadr <dig>
the first described padr proteins are transcriptional repressors for genes encoding phenolic acid decarboxylase  that de-repress padc when phenolic acids are present in toxic amounts  <cit> .
the padr transcription regulator from bacillus subtilis is a prototypical padr-family member protein that binds the padc promoter in the absence of phenolic acid in vitro; binding is lost when exposed to phenolic acids  <cit> .
unlike the prototypical padr, the padr family transcription regulators apha  <cit> , lmrr  <cit> , and bcpadr  <cit>  from vibrio cholerae, lactococcus lactis, and bacillus cereus, respectively, are involved in the regulation of virulence and antibiotic efflux mechanisms.
the prototypical padr and the padr-like transcription regulator apha are within a subfamily of padr proteins  which contain multiple α-helices in the c-terminal domain  <cit> .
another, less studied subfamily of padr family proteins , contains a single α-helix in the c-terminal effector binding/oligomerization domain  <cit> .
the padr-s <dig> proteins, which include the bcpadrs  <cit>  and lmrr  <cit> , have been structurally characterized and are involved in multiple drug recognition.
the bc <dig> gene product, bcpadr <dig>  was upregulated  <dig> -fold in the presence of enterocin treatment in b. cereus atcc <dig> when compared to an untreated control  <cit> .
this padr-like protein binds its own promoter and that of the gene bc <dig>  which encodes a membrane protein predicted to be involved in enterocin as- <dig> resistance  <cit> .
binding of bcpadr <dig> to the predicted promoter region was not affected by the addition of as- <dig> in vitro  <cit> .
the padr-like family protein of l. lactis, lmrr, binds the promoter region of an abc-type multidrug transporter, lmrcd, and interacts with the compound hoechst  <dig> and the antibiotic daunomycin  <cit> .
the crystal structure of apo-lmrr revealed a hydrophobic pore between α <dig> of the dimer mates  <cit> .
additional structures of lmrr bound to hoechst  <dig> and daunomycin, separately, demonstrated that this pore is integral to inhibitor interaction  <cit> .
the conformational change instigated at α <dig> is predicted to interfere with dna binding due to an increase in distance between α <dig> of the dimer mates  <cit> .
this hydrophobic pore is not present in bcpadr structures determined to date.
the genome of hypervirulent c. difficile r <dig> contains the protein coding sequence for three padr-like family proteins .
the function of cdpadr <dig> is of interest due, in part, to its similarity to previously described bcpadrs and lmrr and the response of these transcription regulators to multiple inhibitors.
importantly, differential expression studies have linked cdpadr <dig> and a homologue from historical c. difficile strain  <dig>  to regulatory networks that allow c. difficile to efficiently respond to environmental changes and, thus, survive within a host.
this response is not necessarily due to direct interaction with stressors, but may be part of an overall regulatory cascade.
germination of c. difficile strain  <dig> endospores lead to the differential expression of  <dig> different transcriptional regulators, ~ <dig> % of which were up-regulated as detected by microarray and validated by qrt-pcr  <cit> .
included in this list of differentially expressed transcription regulators is the cdpadr <dig> homologue cd630_ <dig>  which was  <dig> -fold up-regulated during germination  <cit> .
this suggests that the expression of one or more of these proteins required to bring an endospore out of dormancy may be regulated by cd630_ <dig>  another study linked the differential expression of this cdpadr <dig> homologue to acid and alkali shock, oxygen exposure, and subinhibitory concentrations of metronidazole  as detected by microarray analyses in c. difficile strain  <dig>  <cit> .
herein, we investigated the padr-s <dig> protein from c. difficile strain r <dig>  cdpadr <dig>  in this paper, we report the crystallization and x-ray crystal structure of cdpadr <dig> at  <dig>  Å resolution.
we also demonstrate cdpadr <dig> binding to its own gene promoter in a manner conducive to autoregulation.
additionally, we show that cdpadr <dig> binds the promoters of three additional regulatory signaling proteins and that a cdpadr <dig> binding motif is present upstream of  <dig> genes in c. difficile r <dig>
methods
protein expression and purification
residues 1- <dig> of cdpadr <dig>  were amplified from gdna using forward primer pr <dig> –eak  and reverse primer pr4-eak  primers, each of which contained a  <dig> nucleotide overhang followed by restriction digestion sites for bamhi  or psti  for digestion and ligation into the expression vector.
cdpadr <dig> was expressed in escherichia coli rosetta™ using the pqe80l  vector system modified to encode a strep ii™-tag on the n-terminus  <cit> .
cdpadr <dig> was isolated by batch purification over streptactin superflow plus resin .
all buffers were prepared according to the manufacturers’ guidelines.
cell lysis, column equilibration, and wash buffer contained  <dig> mm nah2po <dig> and  <dig> mm nacl .
elution buffer contained  <dig> mm nah2po <dig>   <dig> mm nacl, and  <dig>  mm d-desthiobiotin .
subsequent purification of the cdpadr <dig> dimer was accomplished by size exclusion chromatography in buffer containing  <dig> mm tris  and  <dig> mm nacl, using a superdex  <dig> increase 10/ <dig> gl column connected to an Äkta pure  <dig> .
fractions corresponding to a dimer were concentrated using amicon® concentration units  primed with glycerol and buffer exchanged into  <dig> mm tris  and  <dig> mm kcl.
the molecular weight  was determined by coupling sec with multi-angle light scattering  and outputs were analyzed by the astra software .
crystallization of cdpadr1
crystals were initially obtained by vapor diffusion using a mcsg crystallization suite   with a final protein concentration of  <dig>  mg ml- <dig>  crystal growth was optimized at room temperature by hanging drop vapor diffusion with the drops containing  <dig> μl protein solution  and  <dig> μl reservoir solution .
crystals were transferred into drops containing an equal volume of 2x reservoir solution and  <dig> % glycerol for cryoprotection.
crystals in cryosolution were incubated over original well solution for  <dig> min before freezing in a liquid nitrogen gas stream for cryogenic data collection.
data collection and structure determination
x-ray diffraction data were collected using a marmosaic <dig> ccd detector at the stanford synchrotron radiation lightsource  on beam-line bl14- <dig>  the data were processed with xds and xscale  <cit> .
the xds output files were converted to .mtz format using ccp <dig>  <cit> .
the structure of clostridium thermocellum padr-like family protein  was used as the starting model for molecular replacement using phaser-mr  <cit> .
the individual coordinates of the preliminary model were generated in autobuild  <cit> , were refined and rebuilt using the model in coot  <cit>  and any positions with strong densities outside of the model were accounted for.
structure alignments were performed in coot and all structure/alignment figures prepared using pymol  <cit> .
residues 1– <dig>   <dig>  and 107– <dig> were not modeled due to the absence of electron density.
coordinates have been deposited with the protein data bank  with pdb id 5dym.
data collection and refinement statistics are shown in table  <dig> table  <dig> data collection and refinement statistics
r
merge
b
i/σ
r
work
r
free
astatistics for data in the highest resolution shell are given in parentheses
b
r
merge = /Σi
avg
c
r
work = /Σf
obs
d
r
free was calculated using the test set obtained by randomly selecting  <dig> % of the data
construction of cdpadr1w94a
the tryptophan  <dig>  codon  of cdpadr <dig> was converted to alanine  by overlapping pcr  <cit> .
the sequence of forward and reverse primers used to generate the alanine codon substitutions in cdpadr <dig> were 5′-gaaacaagaagcgagatttattaaaaag-3′ and 5′-ctttttaataaatctcgcttcttgtttc-3′, respectively.
the resulting plasmid was confirmed by sequencing, and the resulting protein variant was overexpressed and purified in the same manner as performed for the native cdpadr <dig>
electrophoretic mobility shift assay
double stranded dna fragments for emsa were generated by suspending custom complementary ssdna  in annealing buffer  and heating to  <dig> °c for  <dig> min followed by slowly cooling to room temperature.
dna was quantified with the quant-it™ broad range dna assay and a qubit® fluorimeter .
template dilutions for emsa stock solutions were dependent on the size of the dna fragment and ranged from  <dig>  μm  to  <dig>  μm .
binding reactions were performed at room temperature.
each reaction mixture contained  <dig> mm tris ph  <dig> ,  <dig> mm kcl,  <dig>  % glycerol,  <dig> mm mgcl <dig>   <dig> mm dtt, and  <dig> μg ml- <dig> heparin.
heparin concentration was increased to  <dig> μg ml- <dig> for competition studies.
a 1: <dig> dilution of dna stock was added to all reactions and a cdpadr <dig> concentration  <dig> -40-fold greater than that of final dna concentration was added to start the binding reaction.
a protein-free control was also included.
emsas were performed in  <dig> % polyacrylamide gels and tb running buffer  at  <dig> v and 20– <dig> ma with run time ranging from  <dig> min  to  <dig> min .
gels were stained with sybr® gold stain .
image coloration was inverted for ease of viewing.
a list of oligonucleotides examined, including location on the genome, sequences, and gc content  can be found in additional file 6: table s <dig>
motif analysis
glam <dig> was utilized to find a representative cdpadr <dig> motif  <cit> .
the sequence surrounding boxes  <dig> &  <dig>  and boxes  <dig> &  <dig>  were used as input sequences for the glam <dig> analysis.
the glam <dig> search was done using the default parameters and allowed the motif sites to be on either the plus or minus strand.
the direct glam <dig> output was used as the input for glam <dig> scan using the c. difficile r <dig> genome.
motifs were allowable on either the minus or plus strand of the genome and  <dig> alignments were allowed.
the identified motifs were then mapped onto the c. difficile r <dig> genome sequence in geneious v <dig>  <cit> .
the motifs were then manually curated to determine whether they were located within an open reading frame, an intergenic promoter region or between convergent genes.
RESULTS
crystal structure of recombinant cdpadr1
cdpadr <dig> shares  <dig> % amino acid sequence identity with the padr-like transcription regulator, cd630_ <dig>  in the historical c. difficile strain  <dig> , both of which were differentially expressed under conditions of environmental stress  <cit> .
cdpadr <dig> crystallized in space group p <dig> and, following x-ray data collection, the structure was solved by molecular replacement using the padr family protein from c. thermocellum  as a search model .
ctpadr and cdpadr <dig> share  <dig> % amino acid sequence identity  and, based on 3d prediction programs  <cit> , were expected to have high structural similarity .
the model was refined to a final crystallographic r-factor of  <dig>  %  .fig.
<dig> amino acid sequence alignment of cdpadr <dig> from clostridium difficile r <dig>  and  <dig>  with structural homologues listed by accession number as follows: 4esb  from bacillus cereus atcc  <dig>  <cit> , 4esf  from b. cereus atcc  <dig>  <cit> , 3f8b  from lactococcus lactis mg <dig>  <cit> , and 1xma  from clostridium thermocellum .
cdpadr <dig> and cd630_ <dig> share  <dig> % amino acid sequence identity.
conserved residues are shaded black.
alpha helices are indicated by black bars and β-sheets are indicated with black arrows.
the highly conserved w residue is demarcated with a black asterisk
one molecule of cdpadr <dig> was present in the asymmetric unit and consists of an n-terminal winged helix-turn-helix  domain  and a single α-helical c-terminal domain  .
this small c-terminal domain places cdpadr <dig> in the padr-s <dig> subfamily of padr transcriptional regulators described previously  <cit> .
cdpadr <dig> forms a dimer with a 2-fold crystallographic axis of symmetry , similar to the bcpadrs  and lmrr , both of which are padr-s <dig> family proteins.
the dimeric state of cdpadr <dig> is retained in solution as determined by size exclusion chromatography .
the recognition helices  are positioned ~ <dig> Å apart  consistent with symmetrical binding to two “half-sites” approximately  <dig> bp in length  <cit> .
dimerization of cdpadr <dig> buries approximately  <dig> Å <dig> solvent-accessible surface area  of the approximately  <dig> Å <dig> total solvent-accessible area per subunit  <cit> .
residues on helices α <dig>  α <dig>  and α <dig> that interact to form the cdpadr <dig> dimer interface are conserved across structural homologues .
the rmsd values for the cdpadr <dig> structural homologues bcpadr <dig>  bcpadr <dig>  apo-lmrr, lmrr-h <dig>  and lmrr-daunomycin are  <dig>  Å,  <dig>  Å,  <dig>  Å,  <dig>  Å, and  <dig>  Å, respectively  <cit> .fig.
<dig> overall structure of cdpadr <dig>  a ribbon representation of cdpadr <dig> monomer with a rainbow color gradient from the n-terminus  to the c-terminus .
alpha helices and β-sheets are labeled numerically.
the winged helix-turn-helix  dna binding domain is indicated.
b the cdpadr <dig> structure is shown perpendicular to the two-fold axis of symmetry with the dna recognition helices indicated .
the dimer mate is shown in gray.
distance between α3/α3′ was estimated in pymol  <cit> .
another view is shown after a 90° forward rotation which results in a view along the two-fold axis facing α4/α4′ with the conserved w residue
the primary helices involved in dimerization are α <dig> and α <dig>  the amino acid sequence pairwise identities between α <dig> of cdpadr <dig> and bcpadr <dig>  bcpadr <dig>  and lmrr are  <dig> %,  <dig> %, and  <dig> %, respectively.
the amino acid sequence pairwise identity between α <dig> of cdpadr <dig> and bcpadrs  is higher than the identity between α <dig> of cdpadr <dig> and lmrr .
helix α <dig> and α4′of cdpadr <dig> bend toward each other  and interact via a coiled-coil, whereas α <dig> and α4′ of lmrr do not display a significant bending towards each other at the c-terminus .
in addition, lmrr contains fewer residues involved in dimerization at the c-terminus of the helix than cdpadr <dig> and bcpadrs.
cdpadr <dig>  like bcpadrs and ctpadr, has a closed dimeric interface, unlike the hydrophobic pore wherein aromatic drug-interaction occurs in lmrr .
the known structural homologues of cdpadr <dig> contain a conserved w located within residues 91– <dig> in the α <dig> helix region that is predicted to be involved in both dimerization and drug binding  <cit> .
the distance between the conserved w residues in the α <dig> helix dimer mates for cdpadr <dig>  bcpadr <dig> , apo-lmrr , lmrr-h <dig> , and lmrr-daunomycin  was measured using chimera  <cit> .
distances were determined from the centroids of the phenol rings , indole rings , and indole-to-phenol  of the conserved α <dig> w residues.
the p-p, i-i, and i-p distances between cdpadr <dig> w <dig> and w94′ are  <dig>  Å,  <dig>  Å, and  <dig>  Å, respectively.
these distances are similar to those of bcpadr <dig> .
the p-p distance is ~ <dig> Å greater for the apo-lmrr , lmrr-h <dig> , and lmrr-daunomycin  structures when compared to the distance between phenol centroids in cdpadr <dig>  the increased distance between α <dig> and α4′of lmrr allows for aromatic inhibitor interaction via π-stacking between the w <dig> and w96′ residues  <cit> .
the lack of a drug-binding pocket in cdpadr <dig> suggests that any differential expression of the cd <dig> homologue  during mtz exposure would, most likely, be due to a regulatory cascade effect rather than direct interaction of cdpadr <dig> with mtz.
it was suggested that changes in the orientation of α <dig> and α4′in a drug-bound state effects the position of the dna recognition helices, rotating them away from each other  <cit> .
this, presumably, would cause a change in dna-binding.
previous work revealed that lmrr binds two sites within the lmrcd promoter, one region containing the predicted - <dig> and - <dig> sites and the other containing the inverted repeats atgt/acat separated by  <dig> nucleotides and that this is consistent with a “conserved” binding motif among other padr-like regulators with an eight nucleotide linker between the inverted repeats atgt/acat  <cit> .
the recognition helices  are positioned ~ <dig> Å apart in cdpadr <dig> , which is consistent with symmetrical binding to two “half-sites” comprised of inverted repeats ~ <dig> bp apart; it is important to note that this distance does not account for dna secondary structure.
dna binding behavior was explored for cdpadr <dig> to determine if it functions similarly to previously studied padr family transcription regulators and to begin elucidating the regulatory networks of cdpadr <dig> in hypervirulent c. difficile in vitro.fig.
<dig> differences between cdpadr <dig>  bcpadr <dig> , and apo-lmrr .
a superposition of cdpadr <dig> , bcpadr <dig> , and lmrr  in cartoon representation with the conserved w indicated .
b surface representations of cdpadr <dig> and apo-lmrr along the two-fold axis facing α4/α4′ , which highlights the hydrophobic pore in lmrr and the closed dimeric interface of cdpadr1
cdpadr <dig> binding to its own promoter
a  <dig> bp region upstream of cdpadr <dig>  was used in emsa assays to determine if cdpadr <dig> binds its own promoter .
the presence of five bands with differing mobility indicated that protein-dna complexes of varying stoichiometry were produced.
this may be the result of multiple binding sites and/or higher order oligomerization upon dna binding .
increasing the concentration of cdpadr <dig> in the reaction resulted in a variation of the migration pattern until an observed saturation point at the slowest mobility compared to other bands was achieved .
cdpadr <dig> binding to pcdpadr <dig>  is consistent with auto-regulation of its own expression.fig.
<dig> emsas of cdpadr <dig> binding the cdpadr <dig> promoter  and smaller regions containing predicted binding boxes.
a pcdpadr <dig> fragments that were bound by cdpadr <dig> are illustrated in green above and those that were not bound are illustrated in gray below the pcdpadr <dig> sequence.
the predicted - <dig> and - <dig> sites are indicated in blue boxes above the sequence.
b final pcdpadr <dig>  concentration in the reaction was  <dig>  μm.
cdpadr <dig> was  <dig>   <dig>   <dig>  and 40-fold excess over dna.
c-d final pr <dig>  and pr <dig>  concentration in the reactions were  <dig>  μm.
cdpadr <dig> was  <dig>   <dig>   <dig>  and 40-fold excess over dna.
e-f the  <dig> bp pcdpadr <dig> fragment  contains predicted binding boxes  <dig> and  <dig>  separated by  <dig> bp and contains a  <dig> bp overhang on both the 5′ and 3′ ends.
the  <dig> bp pcdpadr <dig> fragment  contains predicted binding boxes  <dig> and  <dig>  and contains  <dig> bp overhangs on both the 5′ and 3′ ends.
final pr <dig>  and pr <dig>  concentration in the reactions were  <dig>  μm.
cdpadr <dig> was  <dig>   <dig>   <dig>  and 8-fold excess over dna.
g final dsdna concentration varied depending on the size of the fragment; the + lane contains 10-fold excess cdpadr <dig> over dna.
for emsa gels b-f the minus  lane contains dna and no cdpadr <dig>  shifted dna-protein complexes are annotated with a black arrow and unbound dna migration is marked with a red arrow
to further define the binding sites for cdpadr <dig> binding to pcdpadr <dig>  palinsight was used to identify inverted repeats within pr <dig> characteristic of those bound by transcriptional regulators containing a hth motif .
two sets of inverted repeats  were identified with a tactagta sequence motif .
a series of smaller dsdna fragments within the  <dig> bp pcdpadr <dig> were designed to test the role of these inverted repeats in cdpadr <dig> binding to pcdpadr <dig> .
a  <dig> bp fragment containing both sets of inverted repeats  showed four shifts of varying stoichiometry similar to that seen for pr <dig> .
however, full saturation, as seen for pr <dig>  was not achieved suggesting that additional space on the dna for higher order oligomerization is needed to see complete shifting to one higher molecular weight complex.
when cdpadr <dig> bound a  <dig> bp fragment that contained only one set of inverted repeats  three shifted complexes were observed .
this is consistent with the loss of a full binding site and additional space on the dna for higher order oligomerization as noted for pr <dig>
we further narrowed cdpadr <dig> binding to two small regions of pcdpadr <dig>  each containing one set of inverted repeats tactagta .
cdpadr <dig> bound the  <dig>  and  <dig> bp  regions of pcdpadr <dig> with a single stoichiometry as visualized using emsa .
additionally, a variety of dsdna fragments representing various sub-regions of the original  <dig> bp pcdpadr <dig>  were examined and, unless the fragment contained the predicted inverted repeats tactagta, no binding was observed .
it was noted that the n11- <dig> spacer region within the inverted repeats was at rich.
to determine whether the at richness contributes to localized bending of the dna that facilitates binding we replaced the ttata in pr <dig> with a gcctg sequence .
indeed, significant binding of cdpadr <dig> to pr <dig> was not observed  suggesting that the at-rich spacer is important for binding.
it should be noted that a fragment containing the at rich portion but lacking the intact tact/agta  was not bound by cdpadr <dig> .
this indicates that the at rich sequence is not the direct binding site for cdpadr <dig>  additionally, varying the length of the spacer between the tact/agta inverted repeat in pr <dig> did not interfere with binding  suggesting that flexibility of the dna region between the inverted repeat rather than the length is more important for cdpadr <dig> binding.
to summarize, cdpadr <dig> binding to pcdpadr <dig> is dependent upon a tact/agta inverted repeat sequence.
two such sequences are present in the  <dig> bp pcdpadr1investigated in this study.
these two inverted repeats are responsible for two sequence-specific interactions between cdpadr <dig> and its promoter that can account for two shifted complexes.
additional shifted complexes may be the result of higher order oligomerization of cdpadr <dig> once bound to dna or a decrease in the constraints on sequence specificity.
although constraints on the spacing between the tact/agta inverted repeats do not appear to be tight, there does appear to be a requirement for at richness within the spacer.
the placement of the inverted repeat within pcdpadr <dig> is consistent with auto regulation.
cdpadr <dig> and the cdpadr <dig> homologue cd630_ <dig> both contain tact/agta with an  <dig> nucleotide spacer  <dig> bp upstream of the open reading frame .
additionally, both promoter regions contain tactagta  <dig> bp upstream of their respective orfs and overlapping the predicted -35/- <dig> promoter region, which suggests a similar binding function for each of these genes to their respective promoters.
cdpadr <dig> binds other gene promoters with the cdpadr <dig> motif
the dsdna fragments containing tactagta from p
cdpadr <dig> were analyzed for conserved binding motif using glam <dig>  <cit> .
glam <dig> was advantageous over meme because it allows for spacing/gaps in motif prediction since spacing between the inverted repeats was not critical for binding.
the best motif was  <dig> bp in length with the sequence gtactatattataagta and was designated cdpadr <dig> motif .
glam2scan results indicated the presence of  <dig> potential motif matches in the c. difficile strain r <dig> genome with scores ranging from  <dig> – <dig> , not including the pcdpadr <dig> sequences used for analysis .
approximately half of these motifs are either situated between two convergent genes or are located within open reading frames .
of those that are located upstream of genes, approximately  <dig> % are upstream of other transcription regulators and other regulatory proteins, such as two-component response regulators, while another ~ <dig> % are upstream of genes involved in transport/efflux and sporulation.
the genes predicted to be involved in transport/efflux are the abc transporter atp-binding proteins cdr20291_ <dig>  _ <dig>  _ <dig>  _ <dig>  and _ <dig> .
two genes predicted to be involved in sporulation also contain the cdpadr <dig> binding motif upstream of the transcription start site, a spore maturation protein  and a spore coat assembly protein  .
over  <dig> % of the predicted binding motifs were indicated to be either upstream genes of “hypothetical proteins”, within open reading frame, or between convergent genes.
exemplar promoters from this list were selected for analysis using emsa to determine binding of cdpadr <dig> to these promoter fragments in vitro .
a  <dig> bp and  <dig> bp dsdna fragment was selected for each promoter region and contained at least one predicted cdpadr <dig> motif .
pr <dig> and pr <dig> contain the cdpadr <dig> motif located  <dig> base pairs upstream of cdr20291_ <dig>  .
pr <dig> and pr <dig> contain the cdpadr <dig> motif located  <dig> base pairs upstream of cdr20291_ <dig>  .
pr <dig> and pr <dig> contain the cdpadr <dig> motif located  <dig> base pairs upstream of cdr20291_ <dig>  .
cdpadr <dig> bound all of the selected promoters in vitro .
the  <dig> bp promoters  yielded two discrete bands.
however, this phenomenon has also been observed on occasion for the short dsdna fragment containing one set of inverted repeats from pcdpadr <dig>  and is not likely to represent multiple binding events to a small dsdna fragment  <cit> .
this binding pattern may be attributable to the presence of small amounts of ssdna, portions of the dsdna with secondary structure, or conformational changes in the dna upon binding in a small subset of the complexes which are more pronounced in shorter dsdna fragments  <cit> .fig.
<dig> example analysis of predicted cdpadr <dig> binding motif  as verified by emsa.
a motif identified by glam <dig> analysis  <cit>  of small dsdna fragments from within the cdpadr <dig> promoter .
b
cdpadr <dig> motifs present in three exemplar promoters compared to the cdpadr <dig> motif.
nucleotides that are in agreement with the reference cdpadr <dig> motif are highlighted.
pr <dig> and pr <dig> are upstream of cdr20291_ <dig> ; pr <dig> and pr <dig> are upstream of cdr20291_ <dig> ; pr <dig> and pr <dig> are upstream of cdr20291_ <dig> .
b emsas of cdpadr <dig> binding  <dig> bp  and  <dig> bp  promoter regions that contain cdpadr <dig> motif.
dna only  reactions contained  <dig> nm either  <dig> or  <dig> bp dsdna from promoters listed above.
reactions containing dsdna and  <dig> μm cdpadr <dig> are noted with a plus  sign.
final dna concentration in both reactions was  <dig> nm.
bound complexes are indicated with black arrows
gene regulatory networks play an integral role in the physiology of microorganisms and their response to ever changing environments  <cit> .
the binding of cdpadr <dig> to the promoters of genes encoding transcription regulators and a dna-binding response regulator, part of a two-component signal transduction system, suggests it may play a role in a gene regulatory network in c. difficile.
the cdpadr <dig> motif overlaps the predicted - <dig> region of cdp <dig> and cdp <dig>  <cit> .
this positioning of a regulatory binding site overlapping the - <dig> region is consistent with repression via abrogation of the sigma factor.
in cdp <dig>  the cdpadr <dig> motif is located approximately  <dig> bp upstream of the predicted - <dig> region  <cit> .
positioning of a regulatory binding site upstream of the -35/- <dig> core promoter elements is typically consistent with a role in activation of the promoter  <cit> .
while additional studies are necessary to determine the biological role of cdpadr <dig> in activation or repression of these promoters, it is notable that cdpadr <dig> is able to bind these promoters and likely participates in a regulatory cascade in response to undetermined stimuli.
cdpadr <dig> binds other promoter regions
additional promoters from the cdpadr <dig> genomic neighborhood were chosen to test for cdpadr <dig> binding based on gene expression studies.
a promoter for a nitric oxide reductase  and a spo0b-associated gtp-binding protein were selected.
nitric oxide reductase has been linked to pathogenesis in other microorganisms  <cit>  and was 2-fold down regulated, along with cdpadr <dig> when compared to the historical c. difficile strain  <dig>  <cit> .
another representative promoter for emsa study from within cdpadr <dig> genome neighborhood is upstream of a gene encoding a spo0b-associated gtp-binding protein  whose homologue was 2-fold down regulated following pig loop infection with the historical strain c. difficile  <dig>  <cit> .
cdpadr <dig> bound pnorv and pobg in vitro .
the migration patterns for pnorv and pobg differ from that of pcdpadr <dig> .
for all promoters examined, slower migrating complexes appeared at increasing protein concentrations, which suggests that cdpadr <dig> binds to multiple sites in the upstream region of the gene.
however, the complexes formed when cdpadr <dig> is incubated with promoters other than its own are smaller and it appears that a level of saturation, wherein only one large complex is formed, is not reached as it is for pcdpadr <dig>  it is well understood that transcription regulators bind a relatively limited set of dna sequences  <cit> , a concept that we explored for cdpadr <dig> and p cdpadr <dig> , as well as a predicted binding motif .
both pnorv and pobg have only one half of the inverted repeat within the cdpadr <dig> motif .
however, it is unclear whether only one half-site is sufficient to initiate binding to these promoters or if perhaps the binding is non-specific and related to local dna structure or at content.
therefore, we examined binding specificity using increased amounts of heparin as a competitor for cdpadr <dig> binding .
when a 4-fold higher concentration of heparin was present in the binding reaction of cdpadr <dig> to pnorv or pobg a shifted complex was no longer detected at 40-fold protein over dsdna .
under the same conditions, cdpadr <dig> still bound its own promoter, though the larger complexes were no longer detected.
that cdpadr <dig>  a small hth dna binding protein, would bind other  <dig> bp predicted promoter regions non-specifically could be explained using the theoretical model termed one-dimensional diffusion, or “sliding”.
during one-dimensional diffusion, the transcriptional regulator searches for specific binding sites along the dna remaining in contact with the dna due to non-specific interactions .
it is, therefore, likely that a more specific level of binding requires the full cdpadr <dig> motif.
so, while cdpadr <dig> does, in fact, bind pnorv and other  <dig> bp at-rich promoters in vitro , no conclusions can be made regarding the regulation of this or any other promoters tested based on emsa alone.
coupled with the recent expression studies, however, in vitro binding assays suggest that further study into the regulation of expression of these genes, especially norv, is warranted.fig.
<dig> emsas of cdpadr <dig> binding the cdpadr <dig> promoter , a
norv promoter , and b
obg promoter .
the predicted - <dig> and - <dig> sites are indicated in blue boxes above the sequence.
orange arrows indicate the inverted repeats tact/agta.
c-e final pcdpadr <dig>  pnorv, and pobg  concentration in the reaction was  <dig>  μm.
cdpadr <dig> was  <dig>   <dig>   <dig>  and 40-fold excess over dsdna.
the minus  lane contains dsdna and no cdpadr <dig>  shifted dna-protein complexes are annotated with a black arrow and unbound dsdna migration is marked with a red arrow.
f for this emsa, the final heparin concentration was 4-fold of the standard emsa concentration used throughout this research.
the + lane contains 40-fold excess cdpadr <dig> over dsdna
role of the conserved w residue in cdpadr <dig> dna binding
it was suggested previously that the conformational changes elicited by drug binding between α4/α4′ could affect dna binding and that a conserved tryptophan  in α <dig> was directly involved in drug binding; an indirect role of this w residue was indicated in dna binding  <cit> .
we examined the effect of this conserved w at residue  <dig>  in cdpadr <dig> on dna binding in vitro .
when w <dig> is replaced with an alanine , the majority of binding along with the higher order complexes observed for cdpadr1wt binding to pcdpadr <dig> are lost .
dimerization was not effected as detected by size exclusion chromatography coupled with multi-angle light scattering detection .
these results suggest that, while the conserved w does not affect dimerization, it does inhibit dna binding in vitro in a way that is not entirely clear while further supporting a role of the conserved w in dna binding.
the suggested mechanism by madoori et al wherein the dna binding helices of lmrr putatively rotate away from each other when the effector-binding/oligomerizatoin domain is perturbed at the conserved w residue is further supported as the mechanism of lowered dna binding affinity by the results presented here.fig.
<dig> emsa of cdpadr1wt and cdpadr1w94a binding pcdpadr <dig>  protein-free controls are indicated with a minus sign .
<dig> bp pcdpadr1dna  was used in emsa to determine binding of cdpadr <dig> to its own promoter at increasing concentrations  of protein
CONCLUSIONS
we have determined the  <dig>  Å resolution crystal structure of cdpadr <dig>  which revealed that it is in the padr-s <dig> subfamily of padr transcriptional regulators with other structurally and functionally characterized padr-like regulators from b. cereus  and l. lactis .
in vitro protein-dna binding experiments demonstrate that cdpadr <dig> binds a region comprised of the inverted repeats tact/agta and an at-rich core, gtactatattataagta, within its own promoter.
these predicted binding sites are present in the cdpadr <dig> homologue cd630_ <dig>  suggesting that these transcription regulators are functional homologues as well.
cdpadr <dig> appears to be part of a hierarchical gene regulatory network in c. difficile.
furthermore, cdpadr <dig> non-specifically associates with longer dna fragments that may facilitate promoter and motif searching.
mutation of the highly conserved w in the α <dig> helical region, which is predicted to be involved in multi-drug recognition and dimerization in lmrr, resulted in alterations of cdpadr <dig> binding to the predicted binding motif, potentially due to tighter constraints on spacing of the inverted repeats as well as a loss of higher order oligomerization.
complementary in vivo studies of cdpadr <dig> will allow for a better understanding of its regulatory network.
additional files
additional file 1: figure s <dig>   chromatogram from size exclusion chromatography  run performed on a superdex  <dig> increase 10/ <dig> gl column connected to an Äkta pure  <dig> .
the black line represents the calibration standard mix with molecular weights of standards labeled.
the blue and red lines indicate the elution profile for cdpadr1wt and cdpadr1w94a, respectively .
molar mass versus elution time of cdpadr1wt and cdpadr1w94a from sec  coupled with multi-angle light scattering  detection.
red lines indicate mals signal  and green lines indicate uv detection.
cdpadr1wt and cdpadr1w94a both dimers with molecular weights  of approximately  <dig> and  <dig> kda, respectively .
additional file 2: figure s <dig>  emsa of cdpadr <dig> binding different fragments of pcdpadr <dig>  that contain the inverted repeats tact/agta with  <dig> bp overhang on the 5′ and 3′ end of inverted repeats.
each dsdna contains a different number of nucleotides between tact/agta .
emsas were conducted as described for  <dig> bp and smaller dsdna fragments in the materials and methods.
a complete list of nucleotides tested is available in the table below the emsa gels.
inverted repeats are underlined.
for pr <dig>  the at rich region that was mutated is indicated in bold.
the - lane contains dna only and the + lane contains 10-fold cdpadr <dig> in excess over dna.
additional file 3: table s <dig>  list of glam2scan  <cit>  results for scan of the clostridium difficile r <dig> genome for cdpadr <dig> motif .
the.xlsx file includes the locus id for the gene nearest the motif ; the gene annotation, if applicable, or the indication that this motif is within an open reading frame  or between two convergent genes ; the genomic placement ; indication of whether the motif is on the leading  or lagging  strand ; the motif sequence generated by glam2scan ; and the score generated by glam2scan that indicates how well the sequence fits the cdpadr <dig> motif .
additional file 4: table s <dig>  list of glam2scan  <cit>  results for scan of the clostridium difficile r <dig> genome for cdpadr <dig> motif  for promoters upstream of genes predicted to be involved in transport/efflux and sporulation.
the .xlsx file includes the locus id for the gene nearest the motif ; the gene annotation ; the genomic placement ; indication of whether the motif is on the leading  or lagging  strand ; the motif sequence generated by glam2scan ; and the score generated by glam2scan that indicates how well the sequence fits the cdpadr <dig> motif .
additional file 5: figure s <dig>  emsa of cdpadr <dig> binding the  <dig> bp fragment  that contains the inverted repeats tact/agta with  <dig> nucleotides in between from within its own promoter.
protein-free controls are indicated with a minus sign .
<dig> bp pcdpadr <dig> dna  was used in a reaction with increasing concentrations of cdpadr <dig> .
additional file 6: table s <dig>  full list of oligonucleotides that were annealed and used in emsa studies with cdpadr <dig>  the .xlsx file includes the arbitrary number assigned to each oligonucleotide ; the genomic placement of the minimum nucleotide ; length in base pairs ; indication of binding  or no binding detected  ; locus tag associated with the gene downstream of the oligonucleotide ; the annotated gene downstream of the oligonucleotide ; and the oligonucleotide sequence 5′ to 3′ .
abbreviations
aalanine
ÅÅngström
bc
bacillus cereus
bcpadr1
bacillus cereus locus id bc4206
bcpadr2
bacillus cereus locus id bce3449
bpbase pairs
ccelsius
ccdcharged coupled device
cd
clostridium difficile
cdp1590promoter for locus id cdr20291_1590
cdp1882promoter for locus id cdr20291_1882
cdp2322promoter for locus id cdr20291_2322
cdpadr1locus id cdr20291_0991
ct
clostridium thermocellum
c-terminalcarboxy-terminal
dnadeoxyribonucleic acid
dsdnadouble stranded deoxyribonucleic acid
dttdithiothreitol
emsaelectrophoretic mobility shift assay
gdnagenomic deoxyribonucleic acid
glam2gapped local alignment of motifs version 2
gtpguanosine triphosphate
h33342hoeschst 33342
hepes4--1-piperazineethanesulfonic acid
hthhelix-turn-helix
ididentification
i-iindole-indole
i-pindole-phenol
kclpotassium chloride
lliter
lmrrlactococcal multidrug resistant transcription regulator
mmolar
malsmulti-angle light scattering
mcsgmidwest center for structural genomics
mememultiple em for motif elicitation
mgcl2magnesium chloride
mmmillimolar
mwmolecular weight
nany nucleotide
naclsodium chloride
nah2po4sodium phosphate
naohsodium hydroxide
norvnitric oxide reductase gene
n-terminalamino-terminal
objgene encoding spo0b
orfopen reading frame
padrphenolic acid decarboxylase regulator
padr-s1subfamily of the padr protein family
padr-s2subfamily of the padr protein family
pcdpadr1promoter of cdpadr <dig>
pdbprotein data bank
pnorvpromoter of norv gene
pobgpromoter of obg gene
p-pphenol-phenol
prpromoter
qrt-pcrquantitative reverse transcriptase polymerase chain reaction
rmsdroot mean square deviation
spo0bsporulation initiation phosphotrasferase b
ssdnasingle stranded deoxyribonucleic acid
tbtris-borate
wtryptophan
whthwinged helix-turn-helix
wtwild-type
μlmicroliter
μmmicromolar

 epidemiological trends indicate clinical acquisition of clostridium difficile as the primary route of human infection by this bacterium. the risk of c. difficile becoming a community-acquired infection is likely to increase without the development of better identification and more effective treatment. the genome of c. difficile has been described as “highly dynamic” based on the prevalence of horizontal gene transfer. the impact of a genome that readily changes in response to environmental stress could be a major indicator of c. difficile pathogenicity. c. difficile produces spores that allow it to be viable for extended periods, even in the presence of antibiotics, which could explain the persistence of this human pathogen during host-to-host transmission and in the hospital environment. transcription factors orchestrate the regulation of survival, proliferation, virulence, and antibiotic resistance mechanisms of human pathogens. as part of our larger goal aimed at elucidating structure and function of transcription regulatory mechanisms involved in virulence and antibiotic resistance of human pathogens, we focused on protein targets from a hypervirulent strain of c. difficile. herein, we present our results on a member of the padr family of transcription regulators that we have named cdpadr the first described padr proteins are transcriptional repressors for genes encoding phenolic acid decarboxylase that de-repress padc when phenolic acids are present in toxic amounts. the padr transcription regulator from bacillus subtilis is a prototypical padr-family member protein that binds the padc promoter in the absence of phenolic acid in vitro; binding is lost when exposed to phenolic acids. unlike the prototypical padr, the padr family transcription regulators apha, lmrr, and bcpadr from vibrio cholerae, lactococcus lactis, and bacillus cereus, respectively, are involved in the regulation of virulence and antibiotic efflux mechanisms. the prototypical padr and the padr-like transcription regulator apha are within a subfamily of padr proteins which contain multiple α-helices in the c-terminal domain. another, less studied subfamily of padr family proteins, contains a single α-helix in the c-terminal effector binding/oligomerization domain. the padr-s proteins, which include the bcpadrs and lmrr, have been structurally characterized and are involved in multiple drug recognition. the bc gene product, bcpadr was upregulated -fold in the presence of enterocin treatment in b. cereus atcc when compared to an untreated control. this padr-like protein binds its own promoter and that of the gene bc which encodes a membrane protein predicted to be involved in enterocin as- resistance. binding of bcpadr to the predicted promoter region was not affected by the addition of as- in vitro. the padr-like family protein of l. lactis, lmrr, binds the promoter region of an abc-type multidrug transporter, lmrcd, and interacts with the compound hoechst and the antibiotic daunomycin. the crystal structure of apo-lmrr revealed a hydrophobic pore between α of the dimer mates. additional structures of lmrr bound to hoechst and daunomycin, separately, demonstrated that this pore is integral to inhibitor interaction. the conformational change instigated at α is predicted to interfere with dna binding due to an increase in distance between α of the dimer mates. this hydrophobic pore is not present in bcpadr structures determined to date. the genome of hypervirulent c. difficile r contains the protein coding sequence for three padr-like family proteins. the function of cdpadr is of interest due, in part, to its similarity to previously described bcpadrs and lmrr and the response of these transcription regulators to multiple inhibitors. importantly, differential expression studies have linked cdpadr and a homologue from historical c. difficile strain to regulatory networks that allow c. difficile to efficiently respond to environmental changes and, thus, survive within a host. this response is not necessarily due to direct interaction with stressors, but may be part of an overall regulatory cascade. germination of c. difficile strain endospores lead to the differential expression of different transcriptional regulators, ~ % of which were up-regulated as detected by microarray and validated by qrt-pcr. included in this list of differentially expressed transcription regulators is the cdpadr homologue cd630_ which was -fold up-regulated during germination. this suggests that the expression of one or more of these proteins required to bring an endospore out of dormancy may be regulated by cd630_ another study linked the differential expression of this cdpadr homologue to acid and alkali shock, oxygen exposure, and subinhibitory concentrations of metronidazole as detected by microarray analyses in c. difficile strain. herein, we investigated the padr-s protein from c. difficile strain r cdpadr in this paper, we report the crystallization and x-ray crystal structure of cdpadr at Å resolution. we also demonstrate cdpadr binding to its own gene promoter in a manner conducive to autoregulation. additionally, we show that cdpadr binds the promoters of three additional regulatory signaling proteins and that a cdpadr binding motif is present upstream of genes in c. difficile r methods protein expression and purification residues 1- of cdpadr were amplified from gdna using forward primer pr –eak and reverse primer pr4-eak primers, each of which contained a nucleotide overhang followed by restriction digestion sites for bamhi or psti for digestion and ligation into the expression vector. cdpadr was expressed in escherichia coli rosetta™ using the pqe80l vector system modified to encode a strep ii™-tag on the n-terminus. cdpadr was isolated by batch purification over streptactin superflow plus resin. all buffers were prepared according to the manufacturers’ guidelines. cell lysis, column equilibration, and wash buffer contained mm nah2po and mm nacl. elution buffer contained mm nah2po mm nacl, and mm d-desthiobiotin. subsequent purification of the cdpadr dimer was accomplished by size exclusion chromatography in buffer containing mm tris and mm nacl, using a superdex increase 10/ gl column connected to an Äkta pure. fractions corresponding to a dimer were concentrated using amicon® concentration units primed with glycerol and buffer exchanged into mm tris and mm kcl. the molecular weight was determined by coupling sec with multi-angle light scattering and outputs were analyzed by the astra software. crystallization of cdpadr1 crystals were initially obtained by vapor diffusion using a mcsg crystallization suite with a final protein concentration of mg ml- crystal growth was optimized at room temperature by hanging drop vapor diffusion with the drops containing μl protein solution and μl reservoir solution. crystals were transferred into drops containing an equal volume of 2x reservoir solution and % glycerol for cryoprotection. crystals in cryosolution were incubated over original well solution for min before freezing in a liquid nitrogen gas stream for cryogenic data collection. data collection and structure determination x-ray diffraction data were collected using a marmosaic ccd detector at the stanford synchrotron radiation lightsource on beam-line bl14- the data were processed with xds and xscale. the xds output files were converted to.mtz format using ccp. the structure of clostridium thermocellum padr-like family protein was used as the starting model for molecular replacement using phaser-mr. the individual coordinates of the preliminary model were generated in autobuild, were refined and rebuilt using the model in coot and any positions with strong densities outside of the model were accounted for. structure alignments were performed in coot and all structure/alignment figures prepared using pymol. residues 1– and 107– were not modeled due to the absence of electron density. coordinates have been deposited with the protein data bank with pdb id 5dym. data collection and refinement statistics are shown in table  table data collection and refinement statistics      r merge b   i/σ   r work   r free astatistics for data in the highest resolution shell are given in parentheses b r merge = /Σi avg c r work = /Σf obs d r free was calculated using the test set obtained by randomly selecting % of the data construction of cdpadr1w94a the tryptophan codon of cdpadr was converted to alanine by overlapping pcr. the sequence of forward and reverse primers used to generate the alanine codon substitutions in cdpadr were 5′-gaaacaagaagcgagatttattaaaaag-3′ and 5′-ctttttaataaatctcgcttcttgtttc-3′, respectively. the resulting plasmid was confirmed by sequencing, and the resulting protein variant was overexpressed and purified in the same manner as performed for the native cdpadr electrophoretic mobility shift assay double stranded dna fragments for emsa were generated by suspending custom complementary ssdna in annealing buffer and heating to °c for min followed by slowly cooling to room temperature. dna was quantified with the quant-it™ broad range dna assay and a qubit® fluorimeter. template dilutions for emsa stock solutions were dependent on the size of the dna fragment and ranged from μm to μm. binding reactions were performed at room temperature. each reaction mixture contained mm tris ph, mm kcl, % glycerol, mm mgcl mm dtt, and μg ml- heparin. heparin concentration was increased to μg ml- for competition studies. a 1: dilution of dna stock was added to all reactions and a cdpadr concentration -40-fold greater than that of final dna concentration was added to start the binding reaction. a protein-free control was also included. emsas were performed in % polyacrylamide gels and tb running buffer at v and 20– ma with run time ranging from min to min. gels were stained with sybr® gold stain. image coloration was inverted for ease of viewing. a list of oligonucleotides examined, including location on the genome, sequences, and gc content can be found in additional file 6: table s motif analysis glam was utilized to find a representative cdpadr motif. the sequence surrounding boxes & and boxes & were used as input sequences for the glam analysis. the glam search was done using the default parameters and allowed the motif sites to be on either the plus or minus strand. the direct glam output was used as the input for glam scan using the c. difficile r genome. motifs were allowable on either the minus or plus strand of the genome and alignments were allowed. the identified motifs were then mapped onto the c. difficile r genome sequence in geneious v. the motifs were then manually curated to determine whether they were located within an open reading frame, an intergenic promoter region or between convergent genes.  crystal structure of recombinant cdpadr1 cdpadr shares % amino acid sequence identity with the padr-like transcription regulator, cd630_ in the historical c. difficile strain, both of which were differentially expressed under conditions of environmental stress. cdpadr crystallized in space group p and, following x-ray data collection, the structure was solved by molecular replacement using the padr family protein from c. thermocellum as a search model. ctpadr and cdpadr share % amino acid sequence identity and, based on 3d prediction programs, were expected to have high structural similarity. the model was refined to a final crystallographic r-factor of %.fig. amino acid sequence alignment of cdpadr from clostridium difficile r and with structural homologues listed by accession number as follows: 4esb from bacillus cereus atcc, 4esf from b. cereus atcc, 3f8b from lactococcus lactis mg, and 1xma from clostridium thermocellum. cdpadr and cd630_ share % amino acid sequence identity. conserved residues are shaded black. alpha helices are indicated by black bars and β-sheets are indicated with black arrows. the highly conserved w residue is demarcated with a black asterisk one molecule of cdpadr was present in the asymmetric unit and consists of an n-terminal winged helix-turn-helix domain and a single α-helical c-terminal domain. this small c-terminal domain places cdpadr in the padr-s subfamily of padr transcriptional regulators described previously. cdpadr forms a dimer with a 2-fold crystallographic axis of symmetry, similar to the bcpadrs and lmrr, both of which are padr-s family proteins. the dimeric state of cdpadr is retained in solution as determined by size exclusion chromatography. the recognition helices are positioned ~ Å apart consistent with symmetrical binding to two “half-sites” approximately bp in length. dimerization of cdpadr buries approximately Å solvent-accessible surface area of the approximately Å total solvent-accessible area per subunit. residues on helices α α and α that interact to form the cdpadr dimer interface are conserved across structural homologues. the rmsd values for the cdpadr structural homologues bcpadr bcpadr apo-lmrr, lmrr-h and lmrr-daunomycin are Å, Å, Å, Å, and Å, respectively.fig. overall structure of cdpadr a ribbon representation of cdpadr monomer with a rainbow color gradient from the n-terminus to the c-terminus. alpha helices and β-sheets are labeled numerically. the winged helix-turn-helix dna binding domain is indicated. b the cdpadr structure is shown perpendicular to the two-fold axis of symmetry with the dna recognition helices indicated. the dimer mate is shown in gray. distance between α3/α3′ was estimated in pymol. another view is shown after a 90° forward rotation which results in a view along the two-fold axis facing α4/α4′ with the conserved w residue the primary helices involved in dimerization are α and α the amino acid sequence pairwise identities between α of cdpadr and bcpadr bcpadr and lmrr are %, %, and %, respectively. the amino acid sequence pairwise identity between α of cdpadr and bcpadrs is higher than the identity between α of cdpadr and lmrr. helix α and α4′of cdpadr bend toward each other and interact via a coiled-coil, whereas α and α4′ of lmrr do not display a significant bending towards each other at the c-terminus. in addition, lmrr contains fewer residues involved in dimerization at the c-terminus of the helix than cdpadr and bcpadrs. cdpadr like bcpadrs and ctpadr, has a closed dimeric interface, unlike the hydrophobic pore wherein aromatic drug-interaction occurs in lmrr. the known structural homologues of cdpadr contain a conserved w located within residues 91– in the α helix region that is predicted to be involved in both dimerization and drug binding. the distance between the conserved w residues in the α helix dimer mates for cdpadr bcpadr, apo-lmrr, lmrr-h, and lmrr-daunomycin was measured using chimera. distances were determined from the centroids of the phenol rings, indole rings, and indole-to-phenol of the conserved α w residues. the p-p, i-i, and i-p distances between cdpadr w and w94′ are Å, Å, and Å, respectively. these distances are similar to those of bcpadr. the p-p distance is ~ Å greater for the apo-lmrr, lmrr-h, and lmrr-daunomycin structures when compared to the distance between phenol centroids in cdpadr the increased distance between α and α4′of lmrr allows for aromatic inhibitor interaction via π-stacking between the w and w96′ residues. the lack of a drug-binding pocket in cdpadr suggests that any differential expression of the cd homologue during mtz exposure would, most likely, be due to a regulatory cascade effect rather than direct interaction of cdpadr with mtz. it was suggested that changes in the orientation of α and α4′in a drug-bound state effects the position of the dna recognition helices, rotating them away from each other. this, presumably, would cause a change in dna-binding. previous work revealed that lmrr binds two sites within the lmrcd promoter, one region containing the predicted - and - sites and the other containing the inverted repeats atgt/acat separated by nucleotides and that this is consistent with a “conserved” binding motif among other padr-like regulators with an eight nucleotide linker between the inverted repeats atgt/acat. the recognition helices are positioned ~ Å apart in cdpadr, which is consistent with symmetrical binding to two “half-sites” comprised of inverted repeats ~ bp apart; it is important to note that this distance does not account for dna secondary structure. dna binding behavior was explored for cdpadr to determine if it functions similarly to previously studied padr family transcription regulators and to begin elucidating the regulatory networks of cdpadr in hypervirulent c. difficile in vitro.fig. differences between cdpadr bcpadr, and apo-lmrr. a superposition of cdpadr, bcpadr, and lmrr in cartoon representation with the conserved w indicated. b surface representations of cdpadr and apo-lmrr along the two-fold axis facing α4/α4′, which highlights the hydrophobic pore in lmrr and the closed dimeric interface of cdpadr1 cdpadr binding to its own promoter a bp region upstream of cdpadr was used in emsa assays to determine if cdpadr binds its own promoter. the presence of five bands with differing mobility indicated that protein-dna complexes of varying stoichiometry were produced. this may be the result of multiple binding sites and/or higher order oligomerization upon dna binding. increasing the concentration of cdpadr in the reaction resulted in a variation of the migration pattern until an observed saturation point at the slowest mobility compared to other bands was achieved. cdpadr binding to pcdpadr is consistent with auto-regulation of its own expression.fig. emsas of cdpadr binding the cdpadr promoter and smaller regions containing predicted binding boxes. a pcdpadr fragments that were bound by cdpadr are illustrated in green above and those that were not bound are illustrated in gray below the pcdpadr sequence. the predicted - and - sites are indicated in blue boxes above the sequence. b final pcdpadr concentration in the reaction was μm. cdpadr was and 40-fold excess over dna. c-d final pr and pr concentration in the reactions were μm. cdpadr was and 40-fold excess over dna. e-f the bp pcdpadr fragment contains predicted binding boxes and separated by bp and contains a bp overhang on both the 5′ and 3′ ends. the bp pcdpadr fragment contains predicted binding boxes and and contains bp overhangs on both the 5′ and 3′ ends. final pr and pr concentration in the reactions were μm. cdpadr was and 8-fold excess over dna. g final dsdna concentration varied depending on the size of the fragment; the + lane contains 10-fold excess cdpadr over dna. for emsa gels b-f the minus lane contains dna and no cdpadr shifted dna-protein complexes are annotated with a black arrow and unbound dna migration is marked with a red arrow to further define the binding sites for cdpadr binding to pcdpadr palinsight was used to identify inverted repeats within pr characteristic of those bound by transcriptional regulators containing a hth motif. two sets of inverted repeats were identified with a tactagta sequence motif. a series of smaller dsdna fragments within the bp pcdpadr were designed to test the role of these inverted repeats in cdpadr binding to pcdpadr. a bp fragment containing both sets of inverted repeats showed four shifts of varying stoichiometry similar to that seen for pr. however, full saturation, as seen for pr was not achieved suggesting that additional space on the dna for higher order oligomerization is needed to see complete shifting to one higher molecular weight complex. when cdpadr bound a bp fragment that contained only one set of inverted repeats three shifted complexes were observed. this is consistent with the loss of a full binding site and additional space on the dna for higher order oligomerization as noted for pr we further narrowed cdpadr binding to two small regions of pcdpadr each containing one set of inverted repeats tactagta. cdpadr bound the and bp regions of pcdpadr with a single stoichiometry as visualized using emsa. additionally, a variety of dsdna fragments representing various sub-regions of the original bp pcdpadr were examined and, unless the fragment contained the predicted inverted repeats tactagta, no binding was observed. it was noted that the n11- spacer region within the inverted repeats was at rich. to determine whether the at richness contributes to localized bending of the dna that facilitates binding we replaced the ttata in pr with a gcctg sequence. indeed, significant binding of cdpadr to pr was not observed suggesting that the at-rich spacer is important for binding. it should be noted that a fragment containing the at rich portion but lacking the intact tact/agta was not bound by cdpadr. this indicates that the at rich sequence is not the direct binding site for cdpadr additionally, varying the length of the spacer between the tact/agta inverted repeat in pr did not interfere with binding suggesting that flexibility of the dna region between the inverted repeat rather than the length is more important for cdpadr binding. to summarize, cdpadr binding to pcdpadr is dependent upon a tact/agta inverted repeat sequence. two such sequences are present in the bp pcdpadr1investigated in this study. these two inverted repeats are responsible for two sequence-specific interactions between cdpadr and its promoter that can account for two shifted complexes. additional shifted complexes may be the result of higher order oligomerization of cdpadr once bound to dna or a decrease in the constraints on sequence specificity. although constraints on the spacing between the tact/agta inverted repeats do not appear to be tight, there does appear to be a requirement for at richness within the spacer. the placement of the inverted repeat within pcdpadr is consistent with auto regulation. cdpadr and the cdpadr homologue cd630_ both contain tact/agta with an nucleotide spacer bp upstream of the open reading frame. additionally, both promoter regions contain tactagta bp upstream of their respective orfs and overlapping the predicted -35/- promoter region, which suggests a similar binding function for each of these genes to their respective promoters. cdpadr binds other gene promoters with the cdpadr motif the dsdna fragments containing tactagta from p cdpadr were analyzed for conserved binding motif using glam. glam was advantageous over meme because it allows for spacing/gaps in motif prediction since spacing between the inverted repeats was not critical for binding. the best motif was bp in length with the sequence gtactatattataagta and was designated cdpadr motif. glam2scan results indicated the presence of potential motif matches in the c. difficile strain r genome with scores ranging from –, not including the pcdpadr sequences used for analysis. approximately half of these motifs are either situated between two convergent genes or are located within open reading frames. of those that are located upstream of genes, approximately % are upstream of other transcription regulators and other regulatory proteins, such as two-component response regulators, while another ~ % are upstream of genes involved in transport/efflux and sporulation. the genes predicted to be involved in transport/efflux are the abc transporter atp-binding proteins cdr20291_ _ _ _ and _. two genes predicted to be involved in sporulation also contain the cdpadr binding motif upstream of the transcription start site, a spore maturation protein and a spore coat assembly protein. over % of the predicted binding motifs were indicated to be either upstream genes of “hypothetical proteins”, within open reading frame, or between convergent genes. exemplar promoters from this list were selected for analysis using emsa to determine binding of cdpadr to these promoter fragments in vitro. a bp and bp dsdna fragment was selected for each promoter region and contained at least one predicted cdpadr motif. pr and pr contain the cdpadr motif located base pairs upstream of cdr20291_. pr and pr contain the cdpadr motif located base pairs upstream of cdr20291_. pr and pr contain the cdpadr motif located base pairs upstream of cdr20291_. cdpadr bound all of the selected promoters in vitro. the bp promoters yielded two discrete bands. however, this phenomenon has also been observed on occasion for the short dsdna fragment containing one set of inverted repeats from pcdpadr and is not likely to represent multiple binding events to a small dsdna fragment. this binding pattern may be attributable to the presence of small amounts of ssdna, portions of the dsdna with secondary structure, or conformational changes in the dna upon binding in a small subset of the complexes which are more pronounced in shorter dsdna fragments.fig. example analysis of predicted cdpadr binding motif as verified by emsa. a motif identified by glam analysis of small dsdna fragments from within the cdpadr promoter. b cdpadr motifs present in three exemplar promoters compared to the cdpadr motif. nucleotides that are in agreement with the reference cdpadr motif are highlighted. pr and pr are upstream of cdr20291_; pr and pr are upstream of cdr20291_; pr and pr are upstream of cdr20291_. b emsas of cdpadr binding bp and bp promoter regions that contain cdpadr motif. dna only reactions contained nm either or bp dsdna from promoters listed above. reactions containing dsdna and μm cdpadr are noted with a plus sign. final dna concentration in both reactions was nm. bound complexes are indicated with black arrows gene regulatory networks play an integral role in the physiology of microorganisms and their response to ever changing environments. the binding of cdpadr to the promoters of genes encoding transcription regulators and a dna-binding response regulator, part of a two-component signal transduction system, suggests it may play a role in a gene regulatory network in c. difficile. the cdpadr motif overlaps the predicted - region of cdp and cdp. this positioning of a regulatory binding site overlapping the - region is consistent with repression via abrogation of the sigma factor. in cdp the cdpadr motif is located approximately bp upstream of the predicted - region. positioning of a regulatory binding site upstream of the -35/- core promoter elements is typically consistent with a role in activation of the promoter. while additional studies are necessary to determine the biological role of cdpadr in activation or repression of these promoters, it is notable that cdpadr is able to bind these promoters and likely participates in a regulatory cascade in response to undetermined stimuli. cdpadr binds other promoter regions additional promoters from the cdpadr genomic neighborhood were chosen to test for cdpadr binding based on gene expression studies. a promoter for a nitric oxide reductase and a spo0b-associated gtp-binding protein were selected. nitric oxide reductase has been linked to pathogenesis in other microorganisms and was 2-fold down regulated, along with cdpadr when compared to the historical c. difficile strain. another representative promoter for emsa study from within cdpadr genome neighborhood is upstream of a gene encoding a spo0b-associated gtp-binding protein whose homologue was 2-fold down regulated following pig loop infection with the historical strain c. difficile. cdpadr bound pnorv and pobg in vitro. the migration patterns for pnorv and pobg differ from that of pcdpadr. for all promoters examined, slower migrating complexes appeared at increasing protein concentrations, which suggests that cdpadr binds to multiple sites in the upstream region of the gene. however, the complexes formed when cdpadr is incubated with promoters other than its own are smaller and it appears that a level of saturation, wherein only one large complex is formed, is not reached as it is for pcdpadr it is well understood that transcription regulators bind a relatively limited set of dna sequences, a concept that we explored for cdpadr and p cdpadr, as well as a predicted binding motif. both pnorv and pobg have only one half of the inverted repeat within the cdpadr motif. however, it is unclear whether only one half-site is sufficient to initiate binding to these promoters or if perhaps the binding is non-specific and related to local dna structure or at content. therefore, we examined binding specificity using increased amounts of heparin as a competitor for cdpadr binding. when a 4-fold higher concentration of heparin was present in the binding reaction of cdpadr to pnorv or pobg a shifted complex was no longer detected at 40-fold protein over dsdna. under the same conditions, cdpadr still bound its own promoter, though the larger complexes were no longer detected. that cdpadr a small hth dna binding protein, would bind other bp predicted promoter regions non-specifically could be explained using the theoretical model termed one-dimensional diffusion, or “sliding”. during one-dimensional diffusion, the transcriptional regulator searches for specific binding sites along the dna remaining in contact with the dna due to non-specific interactions. it is, therefore, likely that a more specific level of binding requires the full cdpadr motif. so, while cdpadr does, in fact, bind pnorv and other bp at-rich promoters in vitro, no conclusions can be made regarding the regulation of this or any other promoters tested based on emsa alone. coupled with the recent expression studies, however, in vitro binding assays suggest that further study into the regulation of expression of these genes, especially norv, is warranted.fig. emsas of cdpadr binding the cdpadr promoter, a norv promoter, and b obg promoter. the predicted - and - sites are indicated in blue boxes above the sequence. orange arrows indicate the inverted repeats tact/agta. c-e final pcdpadr pnorv, and pobg concentration in the reaction was μm. cdpadr was and 40-fold excess over dsdna. the minus lane contains dsdna and no cdpadr shifted dna-protein complexes are annotated with a black arrow and unbound dsdna migration is marked with a red arrow. f for this emsa, the final heparin concentration was 4-fold of the standard emsa concentration used throughout this research. the + lane contains 40-fold excess cdpadr over dsdna role of the conserved w residue in cdpadr dna binding it was suggested previously that the conformational changes elicited by drug binding between α4/α4′ could affect dna binding and that a conserved tryptophan in α was directly involved in drug binding; an indirect role of this w residue was indicated in dna binding. we examined the effect of this conserved w at residue in cdpadr on dna binding in vitro. when w is replaced with an alanine, the majority of binding along with the higher order complexes observed for cdpadr1wt binding to pcdpadr are lost. dimerization was not effected as detected by size exclusion chromatography coupled with multi-angle light scattering detection. these results suggest that, while the conserved w does not affect dimerization, it does inhibit dna binding in vitro in a way that is not entirely clear while further supporting a role of the conserved w in dna binding. the suggested mechanism by madoori et al wherein the dna binding helices of lmrr putatively rotate away from each other when the effector-binding/oligomerizatoin domain is perturbed at the conserved w residue is further supported as the mechanism of lowered dna binding affinity by the results presented here.fig. emsa of cdpadr1wt and cdpadr1w94a binding pcdpadr protein-free controls are indicated with a minus sign. bp pcdpadr1dna was used in emsa to determine binding of cdpadr to its own promoter at increasing concentrations of protein  we have determined the Å resolution crystal structure of cdpadr which revealed that it is in the padr-s subfamily of padr transcriptional regulators with other structurally and functionally characterized padr-like regulators from b. cereus and l. lactis. in vitro protein-dna binding experiments demonstrate that cdpadr binds a region comprised of the inverted repeats tact/agta and an at-rich core, gtactatattataagta, within its own promoter. these predicted binding sites are present in the cdpadr homologue cd630_ suggesting that these transcription regulators are functional homologues as well. cdpadr appears to be part of a hierarchical gene regulatory network in c. difficile. furthermore, cdpadr non-specifically associates with longer dna fragments that may facilitate promoter and motif searching. mutation of the highly conserved w in the α helical region, which is predicted to be involved in multi-drug recognition and dimerization in lmrr, resulted in alterations of cdpadr binding to the predicted binding motif, potentially due to tighter constraints on spacing of the inverted repeats as well as a loss of higher order oligomerization. complementary in vivo studies of cdpadr will allow for a better understanding of its regulatory network. additional files additional file 1: figure s chromatogram from size exclusion chromatography run performed on a superdex increase 10/ gl column connected to an Äkta pure. the black line represents the calibration standard mix with molecular weights of standards labeled. the blue and red lines indicate the elution profile for cdpadr1wt and cdpadr1w94a, respectively. molar mass versus elution time of cdpadr1wt and cdpadr1w94a from sec coupled with multi-angle light scattering detection. red lines indicate mals signal and green lines indicate uv detection. cdpadr1wt and cdpadr1w94a both dimers with molecular weights of approximately and kda, respectively. additional file 2: figure s emsa of cdpadr binding different fragments of pcdpadr that contain the inverted repeats tact/agta with bp overhang on the 5′ and 3′ end of inverted repeats. each dsdna contains a different number of nucleotides between tact/agta. emsas were conducted as described for bp and smaller dsdna fragments in the materials and methods. a complete list of nucleotides tested is available in the table below the emsa gels. inverted repeats are underlined. for pr the at rich region that was mutated is indicated in bold. the - lane contains dna only and the + lane contains 10-fold cdpadr in excess over dna. additional file 3: table s list of glam2scan results for scan of the clostridium difficile r genome for cdpadr motif. the.xlsx file includes the locus id for the gene nearest the motif; the gene annotation, if applicable, or the indication that this motif is within an open reading frame or between two convergent genes; the genomic placement; indication of whether the motif is on the leading or lagging strand; the motif sequence generated by glam2scan; and the score generated by glam2scan that indicates how well the sequence fits the cdpadr motif. additional file 4: table s list of glam2scan results for scan of the clostridium difficile r genome for cdpadr motif for promoters upstream of genes predicted to be involved in transport/efflux and sporulation. the.xlsx file includes the locus id for the gene nearest the motif; the gene annotation; the genomic placement; indication of whether the motif is on the leading or lagging strand; the motif sequence generated by glam2scan; and the score generated by glam2scan that indicates how well the sequence fits the cdpadr motif. additional file 5: figure s emsa of cdpadr binding the bp fragment that contains the inverted repeats tact/agta with nucleotides in between from within its own promoter. protein-free controls are indicated with a minus sign. bp pcdpadr dna was used in a reaction with increasing concentrations of cdpadr. additional file 6: table s full list of oligonucleotides that were annealed and used in emsa studies with cdpadr the.xlsx file includes the arbitrary number assigned to each oligonucleotide; the genomic placement of the minimum nucleotide; length in base pairs; indication of binding or no binding detected; locus tag associated with the gene downstream of the oligonucleotide; the annotated gene downstream of the oligonucleotide; and the oligonucleotide sequence 5′ to 3′. abbreviations aalanine ÅÅngström bc bacillus cereus bcpadr1 bacillus cereus locus id bc4206 bcpadr2 bacillus cereus locus id bce3449 bpbase pairs ccelsius ccdcharged coupled device cd clostridium difficile cdp1590promoter for locus id cdr20291_1590 cdp1882promoter for locus id cdr20291_1882 cdp2322promoter for locus id cdr20291_2322 cdpadr1locus id cdr20291_0991 ct clostridium thermocellum c-terminalcarboxy-terminal dnadeoxyribonucleic acid dsdnadouble stranded deoxyribonucleic acid dttdithiothreitol emsaelectrophoretic mobility shift assay gdnagenomic deoxyribonucleic acid glam2gapped local alignment of motifs version 2 gtpguanosine triphosphate h33342hoeschst 33342 hepes4--1-piperazineethanesulfonic acid hthhelix-turn-helix ididentification i-iindole-indole i-pindole-phenol kclpotassium chloride lliter lmrrlactococcal multidrug resistant transcription regulator mmolar malsmulti-angle light scattering mcsgmidwest center for structural genomics mememultiple em for motif elicitation mgcl2magnesium chloride mmmillimolar mwmolecular weight nany nucleotide naclsodium chloride nah2po4sodium phosphate naohsodium hydroxide norvnitric oxide reductase gene n-terminalamino-terminal objgene encoding spo0b orfopen reading frame padrphenolic acid decarboxylase regulator padr-s1subfamily of the padr protein family padr-s2subfamily of the padr protein family pcdpadr1promoter of cdpadr pdbprotein data bank pnorvpromoter of norv gene pobgpromoter of obg gene p-pphenol-phenol prpromoter qrt-pcrquantitative reverse transcriptase polymerase chain reaction rmsdroot mean square deviation spo0bsporulation initiation phosphotrasferase b ssdnasingle stranded deoxyribonucleic acid tbtris-borate wtryptophan whthwinged helix-turn-helix wtwild-type μlmicroliter μmmicromolar 
the crystal structure of cdpadr was determined to Å resolution, which revealed that it belongs to the padr-s subfamily of padr transcriptional regulators. cdpadr binds its own promoter and other promoter regions from within the c. difficile r genome. dna binding experiments demonstrated that cdpadr binds a region comprised of inverted repeats and an at-rich core with the predicted specific binding motif, gtactatattataagta, within its own promoter that is also present in other regions in the c. difficile r genome. our results indicate that cdpadr binds a region within its own promoter consisting of the binding motif gtactatattataagta and seems to associate non-specifically with longer dna fragments in vitro, which may facilitate promoter and motif searching. this suggests that cdpadr acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence. we examined the structure and function of a gene product with the locus tag cdr20291_ as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent c. difficile strain r cdpadr is genomically positioned near genes that are involved in stress response and virulence. clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. clostridium difficiletranscription regulationbacteriastress responsestructural biologypadrprice family foundationn/an/awest ann h. karr elizabeth a. issue-copyright-statement© the author 2016 mutation of the highly conserved w in α of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other padr-s proteins, resulted in alterations of cdpadr binding to the predicted binding motif, potentially due to loss of higher order oligomerization.
 clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. we examined the structure and function of a gene product with the locus tag cdr20291_ as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent c. difficile strain r cdpadr is genomically positioned near genes that are involved in stress response and virulence. in addition, it was previously reported that cdpadr and a homologue from the historical c. difficile strain were differentially expressed when exposed to stressors, including antibiotics.  the crystal structure of cdpadr was determined to Å resolution, which revealed that it belongs to the padr-s subfamily of padr transcriptional regulators. cdpadr binds its own promoter and other promoter regions from within the c. difficile r genome. dna binding experiments demonstrated that cdpadr binds a region comprised of inverted repeats and an at-rich core with the predicted specific binding motif, gtactatattataagta, within its own promoter that is also present in other regions in the c. difficile r genome. mutation of the highly conserved w in α of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other padr-s proteins, resulted in alterations of cdpadr binding to the predicted binding motif, potentially due to loss of higher order oligomerization.  our results indicate that cdpadr binds a region within its own promoter consisting of the binding motif gtactatattataagta and seems to associate non-specifically with longer dna fragments in vitro, which may facilitate promoter and motif searching. this suggests that cdpadr acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords clostridium difficiletranscription regulationbacteriastress responsestructural biologypadrprice family foundationn/an/awest ann h. karr elizabeth a. issue-copyright-statement© the author 2016 