in all domains of life , nucleotide - based second messengers allow a rapid integration of external and internal signals into fine - tuned regulatory pathways that control cellular responses to changing conditions . as a unifying theme , a basic second messenger control module consists of two distinct enzymes for synthesis and degradation of the second messenger and a nucleotide sensor that , upon ligand binding , interacts with a target to produce a cellular output ( hengge , 2009 ) .
3 , 5-cyclic diguanylic acid ( c - di - gmp ) , which is not produced in archaea or eukaryotes , was first discovered as an allosteric effector of cellulose synthase in gluconacetobacter xylinus and is now recognized as one of the most important and widespread second messengers in bacteria .
c - di - gmp is synthesized from two molecules of gtp by diguanylate cyclases ( dgcs ) , which are characterized by active site ggdef motifs ( a - site ) ( paul et  al . , 2004 ; chan et  al . ,
the majority of active dgcs also carry a so - called inhibitory or i - site motif , rxxd , which is involved in feedback inhibition ( christen et  al .
, 2006 ; schirmer and jenal , 2009 ) . specific phosphodiesterases ( pdes ) , which harbor eal or hd - gyp domains , degrade the cyclic dinucleotide ( schmidt et  al . , 2005 ; christen et  al . , 2005 ;
the enzymatically active domains involved in c - di - gmp turnover are often associated with diverse sensory domains , thus enabling cells to adjust second messenger levels in response to different environmental stimuli ( hengge , 2009 ) .
the binding of c - di - gmp to effector proteins impacts diverse processes such as adhesion , virulence , motility , and biofilm formation in unicellular , flagellated bacteria ( rmling et  al . ,
the known c - di - gmp - binding motifs of these proteins are limited but include degenerate ggdef domain proteins carrying i - site motifs ( duerig et  al . , 2009 ;
, 2007b ; petters et  al . , 2012 ) , inactive eal domain receptors ( navarro et  al . , 2009 ;
2009 ) , and pilz domain - containing proteins ( amikam and galperin , 2006 ) .
transcription factors that sense c - di - gmp lack these common c - di - gmp - binding motifs and thus must be identified experimentally .
the sparse list of known c - di - gmp - responsive transcriptional regulators includes the tetr - like activator ltma from mycobacterium smegmatis ( li and he , 2012 ) , the crp - fnr - like transcription factor clp from xanthomonas ( chin et  al . , 2010 ; leduc and roberts , 2009 ) , bcam1349 from burkholderia ( fazli et  al . , 2011
) , the ntrc - type protein fleq from pseudomonas aeruginosa ( baraquet and harwood , 2013 ) , and vpsr from vibrio cholerae ( srivastava et  al . , 2011 ) .
the only c - di - gmp - responsive transcription factor for which structural information is available and hence c - di - gmp binding is understood is vpst , which is a member of the well - studied fixj - luxr - csgd family of response regulators .
the vpst structure revealed a characteristic response regulator fold and a w(f / l / m)(t / s)r c - di - gmp - binding motif ( krasteva et  al . , 2010 ) .
notably , in all known structures of c - di - gmp - binding effector proteins or enzymes , the c - di - gmp is bound either as a monomer or intercalated dimer .
biophysical studies suggest the possibility of higher order oligomeric forms of c - di - gmp , but they have yet to be observed in any biological context ( gentner et  al . , 2012 ) .
while the roles played by c - di - gmp in controlling cellular processes in unicellular bacteria are becoming clear , the function(s ) of c - di - gmp in multicellular , nonmotile bacteria such as streptomyces are unknown .
the complex streptomyces life cycle involves two distinct filamentous cell forms : the growing or vegetative hyphae and the reproductive or aerial hyphae , which differentiate into exospores for dispersion through a massive synchronous septation event ( flrdh and buttner , 2009 ) . in the model species streptomyces venezuelae
, there are three ggdef proteins , two proteins with hd - gyp domains , and five proteins containing both a ggdef and an eal domain ( figure  1a ) . altered expression of the ggdef proteins , cdga and cdgb , and deletions of the eal proteins , rmda and rmdb , have a significant impact on streptomyces growth progression , suggesting that c - di - gmp plays a role in controlling developmental processes in multicellular bacteria ( den hengst et  al . , 2010 ;
interestingly , cdga and cdgb have recently been identified as direct regulatory targets of the developmental master regulator bldd ( den hengst et  al . , 2010 ; tran et  al . ,
mutations in the bld loci block the formation of aerial hyphae , resulting in a  bald
 phenotype , and also affect the production of antibiotics ( mccormick and flrdh , 2012 ) .
bldd sits at the top of the regulatory cascade controlling development , serving to repress expression of sporulation genes during vegetative growth ( den hengst et  al . , 2010 ) .
in streptomyces coelicolor , bldd controls the expression of at least 167 genes , including 42 genes ( 25% of the regulon ) that encode regulatory proteins ( elliot et  al . , 2001 ; den hengst et  al . ,
are many genes known to play critical roles in streptomyces development , including other bld regulators ( e.g. , blda , bldc , bldh / adpa , bldm , and bldn ) , several whi ( white ) regulators required for the differentiation of aerial hyphae into spores ( e.g. , whig and whib ) , and genes encoding critical components of the cell division and chromosome segregation machineries such as ftsz , ssga , ssgb , and the dna translocase sffa ( den hengst et  al . , 2010 ; mccormick , 2009 ) .
we show that bldd is a c - di - gmp - binding effector protein , thus revealing a link between c - di - gmp signaling and the development of multicellular bacteria .
specifically , structural and biochemical analyses show that the second messenger c - di - gmp activates bldd dna binding by driving a unique form of dimerization that is mediated by a tetrameric form of c - di - gmp .
the c - di - gmp tetramer performs its oligomerization function by adjoining two bldd c - terminal domain ( ctd ) protomers , the polypeptide chains of which are separated by 10   .
bldd recognizes the c - di - gmp tetramer using a bipartite rxd - x8-rxxd c - di - gmp interaction signature sequence from each subunit .
thus , tetrameric c - di - gmp acts as a small - molecule dimerizing agent that controls the dna - binding activity of bldd , leading to repression of the bldd regulon of sporulation genes during vegetative growth , thereby controlling the hypha - to - spore transition in multicellular bacteria .
to gain insight into the cellular processes controlled by c - di - gmp in streptomycetes , we overexpressed either the active dgc cdgb from s.  coelicolor ( tran et  al . , 2011 ) or the active pde yhjh from e.  coli ( pesavento et  al . ,
strikingly , overexpression of both cdgb and yhjh blocked the generation of aerial mycelium by s.  venezuelae ( figure  1b ) .
however , scanning electron micrographs ( sems ) revealed that , whereas overexpression of cdgb blocked development , resulting in a classical bald phenotype , overexpression of the pde yhjh in fact promoted sporulation , but the colonies appeared bald to the naked eye because aerial mycelium formation had been bypassed ( figure  1c ) .
as judged by heat resistance , the spores made by the yhjh overexpression strain were as robust as those of the wild - type ( wt ) ( figure  s1a available online ) .
moreover , overexpression of catalytically inactive versions of yhjh or cdgb had no effect on s.  venezuelae development ( figure  s1b ) .
these data suggest that intracellular levels of c - di - gmp influence the timing of development .
in particular , they suggest that increased c - di - gmp levels delay differentiation , arresting the colonies in the vegetative growth stage , whereas decreased levels of the second messenger accelerate development , favoring sporulation .
s.  venezuelae has no pilz domain - containing proteins , and no putative c - di - gmp - binding effector proteins have so far been identified in the streptomyces genus .
thus , to address the mechanism by which s.  venezuelae senses c - di - gmp to control sporulation , we sought to identify c - di - gmp effector proteins involved in development . to selectively enrich putative c - di - gmp - binding proteins from s.  venezuelae cell extracts
, we performed an affinity pull - down assay using a c - di - gmp capture compound ( nesper et  al . , 2012 ) .
remarkably , the developmental master regulator bldd was repeatedly recovered in our c - di - gmp - based capture compound experiments .
bldd is an 18  kda dna - binding protein ( elliot and leskiw , 1999 ) consisting of two distinct domains connected by a flexible linker ( figure  2a ) .
the n - terminal domain is the dna - binding domain ( dbd ) and has a xenobiotic response element ( xre ) helix - turn - helix ( hth ) dna - binding motif ( kim et  al . , 2006 ) .
the bldd ctd harbors a largely helical fold with no known function ( kim et  al . , 2014 ) . to probe the interaction between bldd and c - di - gmp further and to identify the c - di - gmp - binding domain
, we used differential radial capillary action of ligand assays ( dracala ) ( roelofs et  al . , 2011 ) .
dracala allows the visualization of protein - bound radiolabeled ligand as a concentrated spot after the application of the protein - ligand mixture onto nitrocellulose . using this assay
, we confirmed that full - length ( fl ) bldd ( expressed as an n - terminally his6-tagged protein ) from both s.  venezuelae ( figures 2b and s2b ) and s.  coelicolor ( data not shown ) bind p - labeled c - di - gmp .
importantly , the dracala assays demonstrated that the previously uncharacterized ctd of bldd functions as the c - di - gmp - binding domain ( figure  2b ) .
further , excess unlabeled c - di - gmp , but not gtp , competed with the labeled c - di - gmp for binding to fl bldd and to bldd - ctd .
thus , these data reveal that the ctd of the key developmental regulator bldd is a c - di - gmp - binding domain . using global chromatin immunoprecipitation - microarray analysis ( chip - chip )
, we previously identified the complete bldd regulon in s.  coelicolor , showing that it encompasses 167 transcription units ( den hengst et  al . ,
2010 ) . through meme - based sequence analysis of all the promoter regions directly targeted by bldd , we defined a 13  bp pseudo - palindromic sequence , 5-tnac(n)5gtna-3 , designated the bldd box , which functions as a specific binding sequence for bldd ( den hengst et  al . , 2010 ) .
sequence analysis showed that the bldd box was conserved between s.  coelicolor and s.  venezuelae for most key bldd target promoters ( den hengst et  al . , 2010 ) .
further , bldd from s.  coelicolor and s.  venezuelae contain an identical dbd and differ by only two residues in the dbd - ctd linker and five residues in the ctd , suggesting that bldd function is broadly conserved between the two species .
having shown that bldd binds c - di - gmp , we tested the effect of c - di - gmp on bldd dna binding . radiolabeled s.  venezuelae dna fragments encompassing the promoter regions of two well - characterized bldd target genes , bldm and whig , including the bioinformatically identified bldd box ( figure  2c ) , were used as target dnas in electrophoretic mobility shift assays ( emsas ) ( figure  2d ) . the fixed concentration of bldd used in these assays ( 0.6  m ) was insufficient to elicit a dna mobility shift .
however , the addition of increasing concentrations of c - di - gmp ( 0.251.75  m ) strongly induced bldd binding to both the tested promoter regions ( figure  2d ) . to confirm and extend these results into cells
, we manipulated the levels of c - di - gmp in s.  venezuelae and monitored the effect on bldd binding to the bldm and whig promoters in  vivo using chip - sequencing ( chip - seq ) .
the degree of bldd binding was assayed at a single time point in wt s.  venezuelae and the wt overexpressing either the dgc cdgb or the pde yhjh ( the strains whose phenotypes are described above ) .
consistent with the in  vitro emsa data , overexpression of the dgc enhanced chip - seq peak height at the bldd target promoters relative to the wt control ( figure  2e ) .
conversely , overexpression of the pde lowered chip - seq peak heights at bldd target promoters relative to the wt ( figure  2e ) .
these data demonstrate that c - di - gmp enhances the binding of bldd to the bldd box , stimulating bldd - mediated repression of its target regulon .
thus , it is not bldd , but a bldd-(c - di - gmp ) complex , that serves to turn off sporulation genes during vegetative growth .
the opposing effects of the overexpression of the dgc cdgb and the pde yhjh suggested that high levels of c - di - gmp retard sporulation and low levels of c - di - gmp accelerate sporulation . because the bldd-(c - di - gmp ) complex serves to keep sporulation genes shut off during vegetative growth , loss of bldd
should have a similar effect on streptomyces development as depletion of c - di - gmp levels . to test this hypothesis
strikingly , the bldd null mutant formed small colonies lacking aerial hyphae , but  when examined by sem  even young colonies of the bldd mutant were found to contain spore chains embedded in an excess of extracellular matrix ( figure  3a ) .
heat resistance tests showed the bldd mutant spores were mildly defective ( figure  s1a ) .
by contrast , equivalent young colonies of the wt that were grown and imaged in parallel had not yet developed aerial hyphae or spores ( figure  3b ) .
thus , loss of bldd mimics the effects of overexpressing the pde yhjh ( compare figures 1c and 3a ) .
in addition , overexpression of cdgb had no effect on the phenotype of the bldd mutant ( figure  s2a ) , further supporting the idea that c - di - gmp signals through bldd to control the hypha - to - spore transition . to elucidate the molecular basis for c - di - gmp recognition and binding by the bldd ctd and to gain insight into how this interaction may elicit developmental signaling , we determined structures of the s.  venezuelae bldd ctd ( residues 80166 ) and s.  coelicolor bldd ctd ( residues 80167 ) in complex with c - di - gmp . three s.  venezuelae bldd c - domain-(c - di - gmp ) structures and one s.  coelicolor bldd c - domain-(c - di - gmp ) structure were determined to resolutions of 1.95   , 2.33   , 1.75   , and 2.25   , respectively ( figure  4 ) ( see extended experimental procedures and tables s1 and s2 ) .
the bldd ctd structures are composed of two ( -- ) repeats followed by a short c - terminal helix and are similar to the apo form studied by nmr ( kim et  al . , 2014 ) ( figure  4 ) .
database searches revealed that the bldd ctd harbors a new fold , but reduced stringency revealed that it shows limited structural similarity with winged hth proteins , in particular , the winged hth domain of eukaryotic heat shock factor 1 ( hsf1 ) ( littlefield and nelson , 1999 ) .
the bldd ctd and hsf1 bind c - di - gmp and dna , respectively , but the motifs they employ to interact with their nucleotide ligands are completely different ( figure  s3 ) .
the bldd ctd uses a previously unseen mode of c - di - gmp binding in which two noninteracting ctds are glued together by a c - di - gmp tetramer composed of two interlocked c - di - gmp dimers ( figure  4a ) .
thus , in the bldd ctd-(c - di - gmp ) complex , c - di - gmp functions as a macromolecular dimerizer .
indeed , the closest approach of any c atoms of the two tethered ctds is 10   .
the bldd ctd interacts with c - di - gmp using two contiguous surface motifs , herein called motif 1 and motif 2 , which are located between the two -- repeats .
motif 1 is composed of residues 114116 ( rgd ) and motif 2 is composed of residues 125128 ( rqdd ) ( figure  5a ) .
these motifs are located on a solvent exposed region at one end of each ctd protomer and not within a pocket or cavity ( figures 4a and 5a ) .
the combined motifs from two ctd subunits provide ideal shape and electrostatic complementarity for encasing the unusual cage - like c - di - gmp tetramer ( figures 4a and 4b ) . strikingly , although the bldd ctd is overall acidic ( pi5.0 ) , the c - di - gmp - binding surface between two bldd protomers is electropositive ( figure  4b ) .
in addition to shape and charge complementary , contacts from the arginine and aspartic acid residues within motifs 1 and 2 provide specificity for recognition of guanine cyclic nucleotides ; the multiple interactions effectively exclude binding to adenine cyclic nucleotides .
specifically , motif 2 from each ctd protomer combine to mediate contacts to one intercalated c - di - gmp dimer , asp116 of motif 1 from each protomer combine to mediate contacts to the other c - di - gmp dimer , while arg114 sits centrally and anchors both dimers ( figures 5a and 5b ) .
the guanine bases wedged between the two motif 2 regions are specified by contacts from residues arg125 and asp128 ( figures 5a and 5b ) .
residue asp128 makes two hydrogen bonds to the n1 and the exocyclic n2 atoms of the guanine bases on each end ( top and bottom layers ) of the intercalated dimer , while arg125 flanks the guanines in the center ( middle layers ) of the dimer and makes hydrogen bonds to the guanine o6 and n7 atoms ( figures 5b5d ) .
notably , the stacking interactions between the arg125 side chains are the only direct contacts between the two ctd protomers but are clearly not sufficient to promote bldd dimerization .
arg114 is the only ctd residue that makes contacts to both intercalated c - di - gmp dimers .
arg114 hydrogen bonds to the guanines contacted by asp128 , as well as the o6 atoms of the adjacent guanines of the other c - di - gmp dimer ( figures 5a and 5b ) .
thus , arg114 plays a key role in the recognition and stabilization of this unique c - di - gmp tetramer .
the c - di - gmp dimer bound between the motif 1 regions has fewer contacts and is more exposed ( figure  5a ) .
in addition to contacts from arg114 , asp116 of motif 1 hydrogen bonds to the guanine n1 and n2 atoms in a manner analogous to the contacts from asp128 of motif 2 ( figure  5 ) .
notably , the specific hydrogen bonds from motif 1 and 2 residues to guanine exocyclic o6 and n2 atoms dictate that bldd binds to c - di - gmp but not to c - di - amp , which is missing an exocyclic atom at the 2 position and harbors a hydrogen bond donor rather than an acceptor at the exocyclic 6 position .
bldd motifs 1 ( rxd ) and 2 ( rxxd ) , although similar in sequence to the inhibitory i - site ( rxxd ) , which is involved in product inhibition feedback control of dgc activity ( christen et  al . ,
further , mutagenesis of either bldd motif 1 or motif 2 abolishes c - di - gmp binding in dracala assays ( figure  s2b ) , confirming that , unlike i - site c - di - gmp binding , both motifs 1 and 2 in bldd are required to construct the complete binding site for the tetrameric c - di - gmp complex .
this dual signature sequence is unlike any previously characterized c - di - gmp - binding motif .
moreover , specific binding of the c - di - gmp tetramer requires encasement by two such bipartite motifs from precisely oriented bldd protomers .
while the arginine and aspartic acid residues in bldd motifs 1 and 2 dictate the c - di - gmp - binding arrangement and read the guanine bases , contacts to the c - di - gmp phosphate groups are provided by lys84 from 1 and arg130 from 3 .
further , ile110 makes van der waals interactions and residues asn118 and ser123 hydrogen bond with the guanine bases on the top and bottom layers of the c - di - gmp tetramer ( figure  5a ) .
the combination of the optimally positioned bipartite signature sequences from two bldd protomers exquisitely templates binding of the specific and unusual c - di - gmp tetramer structure .
c - di - gmp is monomeric in solution at physiological concentrations ( gentner et  al . , 2012 ) .
however , intercalated c - di - gmp dimers have been observed in crystal structures of the nucleotide alone and in complexes with effector proteins .
higher order c - di - gmp structures such as tetramers and octamers have thus far only been inferred from nmr and spectroscopic studies and require very high c - di - gmp concentrations ( up to 30  mm ) and monovalent cations ( zhang et  al . , 2006 ) .
these higher order structures are characterized by g - quartet interactions with a centrally bound potassium ion coordinated by four guanines .
there are minimal base contacts and no base stacking interactions in these structures ( figure  s4a ) ( zhang et  al . , 2006 ; gentner et  al . ,
2012 ) . by sharp contrast , the bldd - bound tetrameric c - di - gmp is a tightly packed structure that is not secured by ions .
rather , the c - di - gmp molecules are closely spaced and optimally positioned for interbase pairing , leading to the formation of a multistranded , base - stacked structure with top , middle , and bottom layers ( figures 5d and s4a ) .
there are 12 hydrogen bonds between the two intercalated dimers within the c - di - gmp tetramer , including contacts between the n3 atoms and exocyclic nh2 amides of an adjacent base ( figures 5c and s4b ) .
such contacts could not be formed with c - di - amp due to its lack of an exocyclic nh2 atom .
therefore , in addition to contacts from motifs 1 and 2 , guanine - guanine base hydrogen bonds serve to specify c - di - gmp tetramer binding to bldd .
notably , formation of the c - di - gmp tetramer buries 24% of the total surface area ( buried surface area [ bsa ] ) of the c - di - gmp molecules ( figure s4b ) . by comparison , in most protein oligomers
the bsa between protomers is 15% ( wang et  al . , 2009 ) .
finally , the interface between the intercalated c - di - gmp dimers that forms the tetramer is remarkably complementary in shape ( figure  s4b ) .
thus , the combination of multiple contacts between the c - di - gmp moieties along with its extensive bsa and molecular shape complementarity lead to the creation of a compact and highly specific c - di - gmp tetramer . however , bldd is necessary to stabilize this tetramer and template its formation .
the bldd ctd-(c - di - gmp ) crystal structures reveal that c - di - gmp acts as a  dimerizer  to link two ctd protomers . to examine the effect of c - di - gmp on the oligomeric state of the bldd ctd in solution , we carried out chemical crosslinking and  size exclusion chromatography ( sec ) studies .
chemical crosslinking experiments were performed using disuccinimidyl suberate ( dss ) , which contains amine - reactive n - hydroxysuccinimide esters at both ends of an 11   spacer arm .
this reagent should therefore be able to crosslink even the distantly anchored ctd protomers observed in our ctd-(c - di - gmp ) structures ( figure  4a ) . in the absence of dss , the bldd dbd and ctd migrate on sds - page gels as single bands with the expected monomeric molecular weights of 11 and 12  kda , respectively ( figure  6a ) .
upon incubation with dss , the bldd dbd forms a covalent dimer of 21  kda , consistent with previous biochemical and structural analyses of this domain ( kim et  al . , 2006 ; lee et  al . , 2007a ) , and oligomerization is unaffected by the addition of c - di - gmp ( figure  6a ) .
however , in the presence of c - di - gmp , addition of dss results in the clear formation of covalent ctd dimers ( figure  6a ) . to examine c - di - gmp - induced ctd oligomerization further , we performed sec analyses .
as part of this study , we mutagenized the rgd ( motif 1)-x8-rqdd ( motif 2 ) c - di - gmp - binding signature of the bldd ctd . to retain the charge of these surface residues
, we made the charge - swapped dgr - x8-dqdr ctd mutant , changing the key arg residues within the two motifs to asp , and vice versa .
dracala assays showed that this mutant ctd was unable to bind c - di - gmp ( figure  s2b ) .
sec experiments in the presence of 3  m c - di - gmp showed that the wt ctd forms a dimer , while the mutant ctd is monomeric ( figure  6b ) .
hence , the crosslinking and sec data support our structural finding that c - di - gmp is required for bldd ctd dimerization .
further , we constructed a bldd mutant allele encoding a protein solely defective in c - di - gmp binding ( carrying the dgr - x8-dqdr mutation ) and found that it had no ability to complement a bldd mutant ( figure  s2a ) , confirming that the major in  vivo functions of bldd are indeed mediated by its binding to c - di - gmp .
our structures reveal a unique interaction between the bldd ctd and a c - di - gmp tetramer .
remarkably , every guanine in this complex is read specifically by either bldd arginines , aspartic acids , and/or other guanine bases , suggesting that bldd binds only c - di - gmp , and not other cyclic nucleotides . to test this hypothesis and further probe the c - di - gmp - binding affinity of bldd , we performed fluorescence polarization ( fp ) experiments .
these studies were performed with bldd ctd that was expressed and purified from sf9 insect cells to ensure no c - di - gmp was present , as eukaryotes do not produce c - di - gmp ( extended experimental procedures ) .
the bldd-(c - di - gmp ) structures show that one ribose of each c - di - gmp bound to bldd must be unmodified to permit formation of the bldd-(c - di - gmp ) complex ( figure s5a ) .
hence , for these studies we used the fluoresceinated probe , 2-fluo - ahc - c - di - gmp , which harbors the fluorescein dye on only one ribose ( figure  s5a ) .
these studies revealed bldd ctd bound to 2-fluo - ahc - c - di - gmp , with an apparent kd of 2.5  m  0.6 ( figure  s5b ) .
consistent with our dracala assays ( figure  s2b ) , the dgr - x8-dqdr ctd mutant failed to bind 2-fluo - ahc - c - di - gmp in fp assays ( figure  s5b ) .
wt bldd ctd showed no binding to the identically fluoresceinated c - di - amp tagged molecule , 2-fluo - ahc - c - di - amp ( figure  s5b ) .
next , to ascertain the stoichiometry of c - di - gmp binding in solution , we used an fp - based binding assay . the resulting data ( figure  s5c )
a single inflection point can be fitted at a bldd monomer concentration of 12  m , which equates to a stoichiometry of four c - di - gmp molecules per ctd dimer . however
, careful inspection of the data reveals another potential inflection point at a bldd monomer concentration of 6  m , which would be consistent with an initial binding event of two c - di - gmp molecules per ctd dimer .
this putative initial binding event is also apparent in the equilibrium - binding isotherm yielding an apparent kd of 1.7  m ( figure  s5b ) .
two - step binding is not inconsistent with the structure , but the nearly identical affinities observed for each binding event suggest positive cooperativity .
overall , these data demonstrate unequivocally that c - di - gmp binds the ctd with a stoichiometry of four c - di - gmp molecules per ctd dimer , concordant with our  structures .
these studies also demonstrate that the bldd ctd binds specifically and with high affinity to c - di - gmp but not c - di - amp and that both motifs 1 and 2 are essential for this interaction .
dimeric bldd binds pseudo - palindromic dna sites that contain a 5-tnac(n)5gtna-3 consensus ( den hengst et  al . ,
2010 ) . our data and those of others ( lee et  al . ,
2007a ) show that the bldd dbd alone can dimerize at higher concentrations ( 10  m ) .
consistent with these findings , the crystal structure of the s.  coelicolor bldd dbd revealed a dimer with a small contact interface ( kim et  al . , 2006 ) .
we obtained additional views of the bldd dbd by solving the s.  venezuelae bldd dbd structure to 2.80   resolution .
comparison of these dimers with the dimer from the s.  coelicolor dbd structure showed that , although hydrophobic residues within the c - terminal regions of the dbds make contacts between the subunits in each case , all three dimers take distinct conformations ( figure  s6a ) .
moreover , there is less than 300   bsa per subunit in this dimer , which is far less than the 1000   bsa per subunit typically observed for biologically relevant dimers ( krissinel and henrick , 2007 ) .
these data indicate that the bldd dbd is unlikely to form a stable dna - binding active dimer at physiologically relevant concentrations .
in addition , although the bldd dbd resembles the equivalent dbd of  repressor , previous modeling studies suggested that the bldd dbds would not interact specifically with dna if bldd employed a dna - binding mechanism similar to that utilized by  repressor ( kim et  al . , 2006 ) .
our finding that c - di - gmp binding leads to the formation of a c - di - gmp bridged ctd dimer provides the missing link to this puzzle .
however , how c - di - gmp binding to the ctds is signaled to the dbds to bring about dna binding remained unclear . to deduce the mechanism by which c - di - gmp activates bldd to bind dna , we determined the structure of a bldd-(c - di - gmp)-21-mer dna complex to 4.5   resolution ( extended experimental procedures ; figure  s6b ) . while the low resolution of the structure precludes a detailed analysis , the electron density maps show the overall arrangement of the domains and how the dbds dock onto the dna .
critically , the structure reveals that bldd binding to cognate dna is more similar to the dna binding mode of the xre protein sinr ( lewis et  al . , 1998 ;
2013 ) than to that of the  repressor , as the two bldd dbds are juxtaposed when bldd is bound to dna ( figures 7a and 7b ) .
the dbd - dbd interacting surfaces observed in the bldd - dna complex correspond to the hydrophobic regions near the dbd c terminus that interact in  the apo dbd structures .
however , the interfaces are yet again different , supporting the notion that dbd dimerization is weak and malleable .
such malleability is critical to allow the dimeric bldd hth elements to bind the dna , which is bent by 30. the bldd dbds are tethered to the ctd via a linker that was previously shown to be highly flexible ( kim et  al .
not surprisingly , this linker ( pgttpggaaeppp ; residues 7184 ) is disordered in the bldd-(c - di - gmp)-21-mer structure .
its flexibility is underscored by the different orientation of the two ctds in the structure relative to the dbd - dna complex ( figure  7a ) .
the ctd subunits in the dimer make different interactions that help anchor them in the bldd-(c - di - gmp)-dna crystal ; one of the ctds interacts with a hydrophobic patch on its cognate dbd , while the other ctd makes crystal contacts with a symmetry mate ( figures 7a and s6c ) .
however , the ctds in the fl bldd-(c - di - gmp)-dna structure are dimerized in a manner identical to that observed in our ctd-(c - di - gmp ) structures ( figures 4d , 5a , 7a , and s6c ) .
additional evidence that the linker region between the dbd and ctd is flexible was provided by proteolysis experiments employing endoproteinase glu - c , which cleaves exposed peptide bonds on the carboxyl side of glutamic or aspartic acid residues .
hence , if the bldd linker region is unstructured , glu - c should selectively cleave after bldd residue glu80 .
glu - c proteolysis experiments were carried out on the fl bldd protein , the fl protein with the 21-mer dna present , and the fl protein in the presence of both c - di - gmp and the 21-mer dna . in all three cases bldd
was readily cleaved into two bands , corresponding to the dbd and the ctd ( figure  s6d ) . thus , neither the presence of dna , nor of c - di - gmp and dna , protected bldd from proteolysis , indicating the linker is exposed even in the presence of cognate ligands . therefore ,
the combined data suggest a molecular model for c - di - gmp activation of bldd dna binding in which binding of c - di - gmp leads to the formation of a c - di - gmp - linked bldd ctd dimer ( figure  7c ) .
such ctd dimerization effectively brings the two dbds into proximity , thereby increasing their local concentration to allow germane dbd dimerization on cognate dna ( figure  7c ) .
the inherent flexibility afforded by the dbd - ctd linker allows the dbds to adjust for optimal binding to multiple pseudo - palindromic bldd dna boxes .
the role of c - di - gmp has been studied extensively in unicellular gram - negative bacteria , in which most c - di - gmp - dependent signaling pathways control the transition from a planktonic , motile lifestyle to a surface - associated , sessile lifestyle (  stick or swim  ) . here
we show that the activity of the streptomyces master regulator bldd is also controlled by c - di - gmp , thus bringing the regulatory role of this key second messenger into a new physiological arena , that of differentiation in gram - positive multicellular bacteria .
our studies indicate that c - di - gmp binding to bldd controls the developmental switch between vegetative growth and sporulation .
specifically , we demonstrate that c - di - gmp binding to bldd activates its dna - binding activity , which results in repression of sporulation genes during vegetative growth .
consistent with this , genetic studies revealed that bldd null mutants sporulate precociously , mimicking the effect of overexpressing a c - di - gmp phosphodiesterase .
thus , c - di - gmp signals through bldd to control the hypha - to - spore developmental transition in streptomyces .
few c - di - gmp effector - binding motifs have been identified to date .
2009 ; lee et  al . , 2007b ; petters et  al . , 2012 ) , inactive eal domains ( navarro et  al .
, 2009 ; qi et  al . , 2011 ; newell et  al . ,
structures have shown that c - di - gmp interacts with these motifs as either a monomer or intercalated dimer .
the bldd protein does not contain any previously characterized c - di - gmp effector - binding motifs .
thus , to elucidate the mechanism by which c - di - gmp acts as a switch to turn on the dna - binding activity of bldd , we determined several structures of the bldd ctd complexed to c - di - gmp as well as a 4.5   structure of the bldd-(c - di - gmp)-dna complex .
these structures revealed that bldd interacts with c - di - gmp using a heretofore unseen c - di - gmp binding mode involving a unique c - di - gmp - binding signature sequence consisting of two proximal arginine and aspartic acid containing motifs , motif 1 ( rxd ) and motif 2 ( rxxd ) , separated by eight residues .
remarkably , in this binding mode , a tetrameric form of the c - di - gmp functions as a small - molecule dimerizer to adjoin two noninteracting bldd  protomers .
notably , the identical ctd dimer-(c - di - gmp ) tetramer structure was seen in multiple crystal forms .
finally , binding studies confirmed that the bldd ctd binds c - di - gmp with a stoichiometry of four c - di - gmp molecules to one bldd ctd dimer .
the c - di - gmp tetramer revealed in these structures represents a previously unknown form of this nucleotide second messenger .
indeed , we do not know of any other example in which a signaling molecule can assume different oligomeric states to effect its function .
bldd is present throughout the sporulating actinomycetes ( den hengst et  al . , 2010 ) , including , for example , nitrogen - fixing frankia that live in symbiosis within the root nodules of alder trees , and members of the marine genus salinospora , which have recently emerged as an important source of antibiotics and other medically significant compounds . outside of the genus streptomyces ,
the only actinomycete in which bldd has been investigated is saccharopolyspora erythraea , where bldd directly controls expression of the biosynthetic cluster of the clinically important antibiotic erythromycin ( chng et  al . , 2008 ) .
homologs from across the sporulating actinomycetes share 77%99% sequence identity with s.  venezuelae bldd .
the main region of conservation between these proteins is the n - terminal dna - binding domain , which shares 95%100% identity .
although our bldd-(c - di - gmp)-dna structure is too low resolution to ascribe specific protein - dna contacts , it reveals the location of the hth motif and residues that likely contact the dna .
notably , these amino acids are the most conserved among bldd homologs ( essentially 100% ; figure  s7 ) . by contrast , the ctd regions of bldd proteins are less well conserved ( as low as 48% identity ) .
hence , it is striking , given this low conservation , that the residues that interact with c - di - gmp are strictly conserved ( figure  s7 ) .
the only exception is lys84 , which contacts c - di - gmp phosphate groups .
however , in all bldd homologs this residue is either a lysine or arginine and thus able to make the same electrostatic interaction . of particular note , residues r114 , d116 , r125 , and d128 ( from motifs 1 and 2 ) , which mediate essential specifying contacts with c - di - gmp ,
further , all of the actinomycetes that encode an ortholog of bldd also encode ggdef domain - containing dgcs .
these combined findings indicate that bldd-(c - di - gmp ) is likely to control key developmental processes throughout the sporulating actinomycetes , using tetrameric c - di - gmp as a second messenger .
for a full explanation of the experimental protocols , see extended experimental procedures in supplemental information .
strains and plasmids used are shown in table s3 , and oligonucleotides used are shown in table s4 .
a bldd null mutant ( sv77 ) was constructed by redirect pcr targeting , and the bldd::apr mutant allele was moved into a new wt background by generalized transduction using the s. venezuelae - specific phage sv1 .
transduction of the bldd::apr allele was confirmed by pcr and the strain was named sv74 .
cyclic di - gmp capture compound experiments were performed as described previously ( nesper et  al . , 2012 ) but with the minor modifications described in extended experimental procedures .
briefly , the c - di - gmp capture compound was added to the soluble lysates and , following uv irradiation in the caprobox , magnetic streptavidin beads were added to the reaction .
after incubation the  beads were collected , washed , and boiled in sample buffer , the proteins released were run on sds - pa gels and cut out for mass spectrometry analysis .
the dracala assays used his6-bldd or n - terminally his - tagged domains , which were incubated with 11  nm p - c - di - gmp .
the competition experiments had 266  m cold c - di - gmp or gtp added to the reaction .
dna fragments spanning the bldm ( 158  bp ) and whig ( 151  bp ) promoter regions of s.  venezuelae were generated by pcr and 5 end labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed using 0.6  m his6-bldd and radiolabeled dna ( 8,000 cpm ) as well as 0.5  g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.251.75  m ) were added to the mixture .
the reaction samples were incubated for 20  min at room temperature and then run on 5% polyacrylamide gels .
chromatin immunoprecipitations were performed as described ( bush et  al . , 2013 ) using an anti - bldd polyclonal antibody .
the oligomeric states of bldd and its domains were analyzed via chemical crosslinking using dss in the presence and absence of c - di - gmp ( see the extended experimental procedures ) and visualized on sds - pa gels .
molecular weight analyses using sec experiments were performed with a hiload 16/600 superdex 75 pg column . for detailed descriptions of the protein expression , purification , crystallization , structure determination , and refinement protocols ,
see the extended experimental procedures . extended experimental proceduresbacterial strains , growth conditions , and conjugationsall e.  coli strains used in this study ( table s3 ) were grown in lb medium under aeration at 37c .
e.  coli dh5 was used for plasmid and cosmid propagation and bl21(de3)plyss for protein overexpression .
bw25113 ( datsenko and wanner , 2000 ) containing a  red plasmid , pij790 , was used to create the bldd disruption cosmid and et12567 containing puz8002 ( paget et  al . , 1999 )
s.  venezuelae strains ( table s3 ) were grown at 30c on maltose - yeast extract - malt extract ( mym ) medium ( stuttard , 1982 ) containing 50% tap water ( mym - tap ) and 200  l trace element solution ( kieser et  al . , 2000 ) per 100  ml .
conjugations between e.  coli and s.  venezuelae were carried out as previously described ( bibb et  al .
, 2012).construction of plasmidsthe oligonucleotides used for plasmid constructions are listed in table s4 . for protein overexpression and purification , bldd and its individual domains
were cloned into pet15b resulting in n - terminally his - tagged fl ( full - length ) bldd ( amino acid residues 1 - 166 ) , bldd - dbd ( amino acid residues 1 - 79 ) and bldd - ctd ( amino acid residues 80 - 166 ) .
for overexpression of yhjh in s.  venezuelae , an n - terminally codon optimized variant of yhjh was cloned downstream of the ermep promoter in the bt1 attb site - specific integrative vector pij10257 ( hong et  al . , 2005 ) .
point mutations in bldd were introduced by following the four - primer / two - step pcr protocol ( germer et  al . , 2001 ) .
for complementation analysis the bldd gene carrying its native promoter and the r114d , d116r , r125d and d128r mutations was expressed from the integrative vector pms82.construction of a bldd null mutant derivative of s.  venezuelae and phage transductionthe bldd mutant was generated according to the redirect pcr targeting protocol ( gust et  al . , 2003 , 2004 ) .
the sv-4-h05 cosmid was introduced into e.  coli bw25113 and bldd was replaced with the apramycin - resistance ( apr ) cassette containing orit , which was amplified from pij773 using primers with bldd - specific extensions ( table s4 ) .
the disrupted cosmid was confirmed by restriction and pcr analyses and introduced into e.  coli et12567/puz8002 for conjugation into s.  venezuelae .
a null mutant generated by double crossing over was identified by its apramycin - resistant and kanamycin - sensitive phenotype and named sv77 after confirmation by pcr using test primers listed in table s4 .
the mutant allele bldd::apr was moved into a new wt background by generalized transduction using the s.  venezuelae - specific phage sv1 ( stuttard , 1979 ) . to prepare sv1-lysate ,
10 phage were added to 10 sv77 donor spores in 800  l pre - warmed ( 45c ) soft nutrient agar ( sna ) and poured onto difco nutrient agar plates containing 0.5% glucose , 10  mm mgso4 and 10  mm ca(no3)2 .
the plates were incubated at 30c overnight , then flooded with 2.5  ml difco nutrient broth ( dnb ) and incubated for 3 - 4 
the phage - containing dnb soak - out was harvested and filtered through a 0.45  m filter to eliminate bacterial contamination .
for transduction of the bldd::apr allele , 10 phage particles harvested from the bldd::apr mutant strain sv77 , were mixed with 10 - 10 wt spores and incubated overnight on mym agar at room temperature before overlaying with apramycin for selection .
transduction of the bldd::apr allele was confirmed by pcr using test primers listed in table s4 , and the strain was named sv74.c - di - gmp protein capture experimentscyclic di - gmp capture compound experiments were performed as described previously ( nesper et  al . , 2012 ) with minor modifications .
s.  venezuelae cultures were grown in mym - tap supplemented with trace element solution until late transition phase for 24  hr at 30c and then pelleted by centrifugation for 10  min at 6,000  rpm .
the pellet was resuspended in lysis buffer ( 6.7  mm mes , 6.7  mm hepes , 200  mm nacl , 6.7  mm potassium acetate ( kac ) , ph 7.5 ) containing protease inhibitor and dnase i. cells were put through a french press four times at 18,000 psi and then centrifuged at 100,000 x g for 1  hr . for the capture experiments , the protein concentration of the soluble fraction
was determined using a uv / vis nanodrop spectrophotometer and 400  g protein were mixed with 10  m c - di - gmp capture compound and with 20  l 5 x capture buffer ( 100  mm hepes , 250  mm kac , 50  mm magnesium acetate ( mgac ) , 50% glycerol , ph 7.5 ) .
1  mm c - di - gmp was added to the control reaction and incubated for 30  min prior to capture compound addition .
the reaction volume was adjusted with h2o to 100  l and incubated for 2  hr at 4c in the dark on a rotary wheel .
after uv irradiation for 4  min in a caprobox , 50  l magnetic streptavidin beads and 25  l 5 x wash buffer ( 250  mm tris ph 7.5 , 5  m nacl , 0.1% n - octyl--glucopyranoside ) were added to the reaction and the mixture was incubated for 45  min at 4c on a rotary wheel .
the beads were then collected with a magnet and washed 6 times with 200  l wash buffer .
the beads were resuspended in 20  l sample buffer and run for 10  min on a 15% sds polyacrylamide gel after 10  min incubation at 95c .
the gel was stained using instantblue coomassie stain solution , and a 11  cm gel slice containing all captured proteins was excised for analysis by mass spectrometry.electrophoretic mobility shift assaysdna fragments spanning the bldm ( 158  bp ) and whig ( 151  bp ) promoter regions of s.  venezuelae were generated by pcr using oligonucleotides listed in table s4 and then 5 end - labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed in bandshift buffer ( 10  mm tris ph 7.5 , 1  mm edta , 5% glycerol , 10  mm nacl , 1  mm mgcl2 ) in 20  l reaction mixture containing 0.6  m bldd and radiolabeled dna ( 8,000 c.p.m . ) as well as 0.5  g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.25  1.75  m ) were added to the mixture .
the reaction samples were incubated for 20  min at room temperature followed by electrophoresis on a 5% polyacrylamide gel in 0.5 x tbe ( tris - borate - edta ) buffer at 80v for 105  min .
the gels were dried before being analyzed on a phosphorimager.bldd chip - seq experimentss . 
chromatin immunoprecipitations were performed as previously described ( bush et  al . , 2013 ) , except that an anti - bldd polyclonal antibody was used and pulled down with protein a - sepharose beads . library construction ,
sequencing and chip - seq data analyses were all carried out as previously described ( bush et  al .
, 2013).chemical crosslinking and sds polyacrylamide gel electrophoresisthe his6-bldd - dbd and his6-bldd - ctd proteins were dialyzed into crosslinking buffer ( 100  mm nah2po4 , 150  mm nacl , ph 8) and then incubated at room temperature for 30  min in 20  l reaction samples containing 10  m protein , 1  mm disuccinimidyl suberate ( dss ) in dimethylsulfoxide ( dmso ) , and c - di - gmp as indicated .
the reaction was stopped by adding 50  mm tris ph 8 and incubation for 15  min followed by addition of sds sample buffer and heating to 95c for 10  min .
samples were separated on a 15% sds polyacrylamide gel and visualized by coomassie staining.determination of c - di - gmp binding to proteins by differential radial capillary action of ligand assayradiolabeled c - di - gmp was synthesized in  vitro using [ -p]-gtp and the purified diguanylate cyclase pled as described ( paul et  al . , 2004 ) .
the dracala assays ( roelofs et  al . , 2011 ) were performed using 2  g of his6-bldd or its n - terminally his - tagged domains that were incubated with 11  nm p - c - di - gmp in dgc buffer ( 250  mm nacl , 25  mm tris ph 8 , 10  mm mgcl2 , 5  mm -mercaptoethanol ) . for competition experiments ,
266  m cold c - di - gmp or gtp were added to the reaction . after a 5  min incubation at room temperature ,
5  l of the binding sample were spotted onto nitrocellulose membrane and the dried membranes were analyzed using a phosphorimager.purification , crystallization , and structure determination of s.  venezuelae and s.  coelicolor bldd ctd-(c - di - gmp )  complexesfor structural studies on the ctd , the regions encoding residues 80 - 166 ( s.  venezuelae bldd ) and 80 - 167 ( s.  coelicolor bldd ) were cloned into the pet15b vector and the proteins induced at 37c and purified via ni - nta chromatography .
the his - tags were removed from the proteins used for structural studies by thrombin cleavage .
crystals of the s.  venezuelae bldd ctd-(c - di - gmp ) complex , which assumed the trigonal space group , p32 , were obtained using protein at 30  mg / ml and 1  mm c - di - gmp .
crystals were produced via the hanging drop vapor diffusion method and mixing the complex 1:1 with 28% peg 1500 , 100  mm sodium acetate , ph 5.5 .
the s.  venezuelae bldd ctd contains no methionines and hence for phasing , leu92 and ile135 were substituted with methionines .
semet(l92m / i135 m ) bldd ctd was expressed using the methionine inhibitory pathway and the protein purified and crystallized with c - di - gmp as per the wt ctd .
the selenomethionine - substituted l92m / i135 m protein crystallized in the wild - type protein p32 space group .
a second crystal form of the s.  venezuelae bldd ctd-(c - di - gmp ) complex was grown with protein at 20 - 40  mg / ml and 1  mm c - di - gmp using 20% peg 2000 monomethyl ether , 100  mm mes , ph 6.0 , as a crystallization reagent and took the orthorhombic space group p21212 .
the third crystal form of the s.  venezuelae bldd ctd-(c - di - gmp ) complex was produced with protein at 10  mg / ml and 1  mm c - di - gmp using 1.2  m sodium / potassium phosphate , 50  mm citrate ph 5.6 .
crystals were obtained for the s.  coelicolor bldd ctd-(c - di - gmp ) complex using 25  mg / ml protein , 1  mm c - di - gmp and mixing the complex 1:1 with a reservoir comprised of 1.4  m sodium / potassium phosphate , 100  mm hepes ph 7.5 .
these crystals take the c2221 space group.multiple wavelength anomalous diffraction ( mad ) data were collected for a semet(l92m / i135 m ) s.  venezuelae bldd ctd-(c - di - gmp ) crystal to 2.28   resolution at als ( advanced light source , berkeley , ca , usa ) beamline 8.3.1 ( table s1 ) .
the data were processed using mosflm and the heavy atom substructure was obtained via solve ( terwilliger and berendzen , 1999 ) .
phenix was used for final phasing and density modification ( adams et  al . , 2010 ) .
the crystal contains 12 protein molecules in the asymmetric unit ( asu ) and each of the six dimers is glued together by four c - di - gmp molecules .
the six c - di - gmp complexed dimers are essentially identical ( figure  4d ) .
final refinement was done using a data set collected to 1.95   resolution for a semet(blddl92 m ) ctd-(c - di - gmp ) crystal .
a wt data set was also collected to 2.2   resolution and the structure was identical to the l92 m and l92m / i135 m structures .
the final 1.95   resolution - structure contains residues 84 - 161 for each of the 12 subunits and 24 c - di - gmp molecules ( table s2 ) .
data were collected to 1.75   , 2.25   and 2.33   resolution for the s.  venezuelae and s.  coelicolor bldd ctd-(c - di - gmp ) c2221 forms and the s.  venezuelae bldd ctd-(c - di - gmp ) p21212 crystal form , respectively , and the structures solved by molecular replacement ( mr ) .
the s.  coelicolor bldd ctd and s.  venezuelae bldd ctd c2221 crystal forms contain a ctd dimer and four c - di - gmp molecules in the asu and the s.  venezuelae bldd c - domain-(c - di - gmp ) p21212 crystal form contains 10 subunits ( five dimers ) , and 20 c - di - gmp molecules .
the structures were solved by mr using the program phaser ( mccoy et  al . , 2007 ) .
the topology of every bldd ctd is 1 ( residues 84 - 88)-1 ( residues 89 - 93)-2 ( residues 98 - 113)-2 ( residues 120 - 124)-3 ( residues 128 - 136)-4 ( residues 140 - 149)-5 ( residues 154 - 160).crystallization and structure determination of the s.  venezuelae bldd dbdthe bldd dna binding domain ( bldd dbd ) , encoding residues 1 - 79 , was cloned into pet15b , expressed in e.  coli bl21(de3 ) and the protein purified via ni - nta chromatography .
prior to crystallization , the hexa - his tag was removed via thrombin cleavage and the protein further purified by size exclusion chromatography .
crystals were grown by mixing the protein ( 40  mg / ml ) 1:1 with a reservoir consisting of 35% peg 400 , 0.1  m mgcl2 and 0.1  m tris ph 7.5 .
x - ray intensity data were collected to 2.8   resolution at als beamline 8.3.1 and processed with mosflm .
the rsym and i/(i ) for the data are 11.3% ( 38.4% ) and 11.4 ( 4.3 ) , respectively , where the values in parentheses indicate data from the highest resolution shell .
the structure , which contains 3 subunits in the asu ( 2 subunits form a dimer and crystal symmetry generates a second dimer ) , was solved with phaser using a single s.  coelicolor dbd subunit ( pdb code 2ewt ) as the search model .
the final model contains residues 3 - 71 of each subunit and was refined using phenix to final rwork / rfree values of 23.7%/28.9% , respectively ( adams et  al .
, 2010).crystallization and structure determination of the s.  venezuelae bldd-(c - di - gmp)-21-mer complexcrystals of the full - length ( fl ) s.  venezuelae bldd-(c - di - gmp)-21-mer complex were grown by using protein in which the n - terminal hexa - his tag had been cleaved and incubated with 1  mm c - di - gmp ( final concentration ) .
this bldd-(c - di - gmp ) solution was mixed in a 1:1 molar ratio of bldd dimer to 21-mer dna duplex ( 5-cccctcacgctgcgtgacggg-3 , with the canonical bldd box underlined , annealed to its complementary oligodeoxynucleotide ) for crystallization trials .
crystals were grown by mixing the protein - dna complex 1:1 with the crystallization solution composed of 100  mm sodium citrate tribasic / citric acid ph 4.0 and 200  mm ammonium sulfate .
the crystals take the trigonal space group , p3221 , with a  = b  = 114.0   , c  = 95.2   and contain a bldd dimer-(c - di - gmp)-21-mer duplex in the asu .
data were collected to the limiting resolution of 4.5   . the rsym  = 9.0% ( 86.0% ) and i/(i )  = 5.8 ( 1.8 ) , where the values in parentheses are for the highest resolution shell .
first , bldd dna binding domain - dna complex models were constructed based on the sinr - dna ( pdb code 3zkc ) or  repressor - dna ( pdb code 1lmb ) complex structures and used in the mr program phaser ( mccoy et  al . , 2007 ) .
this solution was then used as a static model with the ctd-(c - di - gmp ) dimeric structure as a search model .
due to its low resolution , the structure was subjected to rigid body refinement only ( rwork / rfree  = 33.0%/38.9% , respectively).proteolysis of fl bldd by endoproteinase glu - cto examine the flexibility of the linker region that connects the bldd dbd and ctd , limited proteolysis experiments were carried out .
specifically , the accessibility of residue glu80 , which is the only acidic residue in the bldd linker region , was determined by the ability of the glu - c protease to cleave after this residue . in these experiments , endo - glu - c ( 100  units / ml ) was a diluted 50 fold into samples of 1 ) fl bldd ( 1  mg / ml ) , 2 ) fl bldd-21-mer dna ( 1  mg / ml protein with 100  m dna ) or 3 ) fl bldd-(c - di - gmp)-21-mer ( 1  mg / ml protein with 100  m 21-mer dna and 1  mm c - di - gmp ) .
the proteolysis buffer was 50  mm sodium phosphate , ph 7.5 and the final protein concentration of each sample was 4  mg / ml .
the samples shown in figure  s6d were taken at a time point of 4  hr .
notably , proteolysis of each sample was identical , demonstrating that ligand binding of neither cognate dna nor c - di - gmp by bldd affect the accessibility of the linker region , which remains flexible and unstructured.size exclusion chromatographic analyses of wild - type bldd ctd and the bldd ctd quadruple mutantsize exclusion chromatography experiments were carried out using a hiload 16/600 superdex 75 pg column .
5  mg of either the wild - type bldd ctd or the quadruple mutant were loaded onto a column that had been pre - equilibrated with 150  mm nacl , 5% glycerol , 20  mm tris hcl ph 7.5 and 3  m c - di - gmp .
the samples were run and eluted with the same c - di - gmp containing buffer .
the elution volume was plotted against a standard curve to determine the relative molecular weights of the samples .
the standard curve was determined using cytochrome c ( 12  kda ) , carbonic anhydrase ( 29  kda ) and albumin ( 66  kda).cryo - scanning electron microscopycryo - sem was performed as previously described ( bush et  al . , 2013).bldd ctd expression and purification from sf9 cellswhen induced in e.  coli bl21(de3 ) at 37c , bldd ctd samples contained little to no c - di - gmp contamination , which was supported by a280/a260 values of the purified protein , which were approximately 1.6 .
however , to ensure that there was no endogenous c - di - gmp present in samples used for binding affinity measurements , the bldd ctd was expressed and purified in sf9 insect cells . for these studies , a gene encoding the same s.  venezuelae bldd ctd region that was expressed in e.  coli was codon optimized for expression in insect cells ( genscript , piscataway , nj , usa ; http://www.genscript.com ) and subcloned into the expression vector f1 , which was transfected into the dh10bac strain for generation of the recombinant bacmid .
rbacmids were then transfected in sf9 insect cells with cellfectin ii and the cells incubated in sf-900 ii sfm for 56  days before harvest .
the supernatant was collected for the p1 viral stock and p2 was amplified for later infection .
sf9 cells expressing bldd ctd were harvested at 72  hr post infection and cells were lysed into 25  mm tris ph 7.5 , 300  mm nacl and 5% glycerol with protease inhibitors .
the protein was purified from the supernatant by ni - nta followed by size exclusion chromatography and was > 90% pure as assessed by sds - page analysis.determination of the affinity , stoichiometry , and specificity of c - di - gmp for sf9-purified bldd ctd by fluorescence  polarizationto measure binding , 2-o-(6-[fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate ( 2-fluo - ahc - c - di - gmp ) , was used as the fluoresceinated ligand .
this molecule is conjugated via a 9 atom spacer to one of the 2 hydroxyl groups of the c - di - gmp , hence meeting the structural requirement for bldd binding that only one 2 hydroxyl group be unmodified and the other available for the interactions observed in the bldd-(c - di - gmp ) structures ( figure  5 ) .
binding was carried out at 25c in a buffer of 150  mm nacl and 25  mm tris - hcl ph 7.5 , which contained 1  nm 2-fluo - ahc - c - di - gmp .
increasing concentrations of bldd ctd were titrated into the reaction mixture to obtain the binding isotherms .
after each addition of protein the reaction sample was incubated for 30  min to ensure equilibrium had been reached .
c - di - gmp binding to bldd appears to have the characteristics of a high affinity / slow binding ligand .
the resulting data were plotted using kaleidagraph and curves were fitted to deduce binding affinities . to determine the binding stoichiometry of the bldd ctd-(c - di - gmp ) complex , the same fp binding conditions were used but the total concentration of c - di - gmp ( c - di - gmp  + 2-fluo - ahc - c - di - gmp ) was 25  m , 10-fold above the kd of 2.5  m ensuring stoichiometric binding .
close inspection of the binding isotherm revealed the possibility that there are two nearly identical binding events ( likely corresponding to the binding of each intercalated c - di - gmp dimer ) .
the graph of the resulting data shows a linear increase in the observed mps until saturation of the binding sites , after which the line is flat .
importantly , the final inflection occurs at a bldd monomer concentrations of 12  m , which , when divided by the concentrations of c - di - gmp ( 25  m ) , indicates a stoichiometry of two ctd protomers per four c - di - gmps .
as anticipated from a purely chemical complementarity - orientated argument , fp studies carried out in the same buffer also revealed that wild - type bldd ctd does not bind to c - di - amp .
in addition , the bldd dgr - x8-dqdr ctd mutant did not bind the c - di - gmp probe .
all e.  coli strains used in this study ( table s3 ) were grown in lb medium under aeration at 37c .
e.  coli dh5 was used for plasmid and cosmid propagation and bl21(de3)plyss for protein overexpression .
bw25113 ( datsenko and wanner , 2000 ) containing a  red plasmid , pij790 , was used to create the bldd disruption cosmid and et12567 containing puz8002 ( paget et  al . , 1999 )
s.  venezuelae strains ( table s3 ) were grown at 30c on maltose - yeast extract - malt extract ( mym ) medium ( stuttard , 1982 ) containing 50% tap water ( mym - tap ) and 200  l trace element solution ( kieser et  al . , 2000 ) per 100  ml .
conjugations between e.  coli and s.  venezuelae were carried out as previously described ( bibb et  al . , 2012 ) .
for protein overexpression and purification , bldd and its individual domains were cloned into pet15b resulting in n - terminally his - tagged fl ( full - length ) bldd ( amino acid residues 1 - 166 ) , bldd - dbd ( amino acid residues 1 - 79 ) and bldd - ctd ( amino acid residues 80 - 166 ) .
for overexpression of yhjh in s.  venezuelae , an n - terminally codon optimized variant of yhjh was cloned downstream of the ermep promoter in the bt1 attb site - specific integrative vector pij10257 ( hong et  al . , 2005 ) .
point mutations in bldd were introduced by following the four - primer / two - step pcr protocol ( germer et  al . , 2001 ) .
for complementation analysis the bldd gene carrying its native promoter and the r114d , d116r , r125d and d128r mutations was expressed from the integrative vector pms82 .
the bldd mutant was generated according to the redirect pcr targeting protocol ( gust et  al .
the sv-4-h05 cosmid was introduced into e.  coli bw25113 and bldd was replaced with the apramycin - resistance ( apr ) cassette containing orit , which was amplified from pij773 using primers with bldd - specific extensions ( table s4 ) .
the disrupted cosmid was confirmed by restriction and pcr analyses and introduced into e.  coli et12567/puz8002 for conjugation into s.  venezuelae .
a null mutant generated by double crossing over was identified by its apramycin - resistant and kanamycin - sensitive phenotype and named sv77 after confirmation by pcr using test primers listed in table s4 .
the mutant allele bldd::apr was moved into a new wt background by generalized transduction using the s.  venezuelae - specific phage sv1 ( stuttard , 1979 ) . to prepare sv1-lysate ,
10 phage were added to 10 sv77 donor spores in 800  l pre - warmed ( 45c ) soft nutrient agar ( sna ) and poured onto difco nutrient agar plates containing 0.5% glucose , 10  mm mgso4 and 10  mm ca(no3)2 .
the plates were incubated at 30c overnight , then flooded with 2.5  ml difco nutrient broth ( dnb ) and incubated for 3 - 4  hr at room temperature . the phage - containing dnb soak - out was harvested and filtered through a 0.45  m filter to eliminate bacterial contamination .
for transduction of the bldd::apr allele , 10 phage particles harvested from the bldd::apr mutant strain sv77 , were mixed with 10 - 10 wt spores and incubated overnight on mym agar at room temperature before overlaying with apramycin for selection .
transduction of the bldd::apr allele was confirmed by pcr using test primers listed in table s4 , and the strain was named sv74 .
cyclic di - gmp capture compound experiments were performed as described previously ( nesper et  al . , 2012 ) with minor modifications .
s.  venezuelae cultures were grown in mym - tap supplemented with trace element solution until late transition phase for 24  hr at 30c and then pelleted by centrifugation for 10  min at 6,000  rpm .
the pellet was resuspended in lysis buffer ( 6.7  mm mes , 6.7  mm hepes , 200  mm nacl , 6.7  mm potassium acetate ( kac ) , ph 7.5 ) containing protease inhibitor and dnase i. cells were put through a french press four times at 18,000 psi and then centrifuged at 100,000 x g for 1  hr .
for the capture experiments , the protein concentration of the soluble fraction was determined using a uv / vis nanodrop spectrophotometer and 400  g protein were mixed with 10  m c - di - gmp capture compound and with 20  l 5 x capture buffer ( 100  mm hepes , 250  mm kac , 50  mm magnesium acetate ( mgac ) , 50% glycerol , ph 7.5 ) . 1  mm c - di - gmp was added to the control reaction and incubated for 30  min prior to capture compound addition .
the reaction volume was adjusted with h2o to 100  l and incubated for 2  hr at 4c in the dark on a rotary wheel .
after uv irradiation for 4  min in a caprobox , 50  l magnetic streptavidin beads and 25  l 5 x wash buffer ( 250  mm tris ph 7.5 , 5  m nacl , 0.1% n - octyl--glucopyranoside ) were added to the reaction and the mixture was incubated for 45  min at 4c on a rotary wheel .
the beads were then collected with a magnet and washed 6 times with 200  l wash buffer .
the beads were resuspended in 20  l sample buffer and run for 10  min on a 15% sds polyacrylamide gel after 10  min incubation at 95c .
the gel was stained using instantblue coomassie stain solution , and a 11  cm gel slice containing all captured proteins was excised for analysis by mass spectrometry .
dna fragments spanning the bldm ( 158  bp ) and whig ( 151  bp ) promoter regions of s.  venezuelae were generated by pcr using oligonucleotides listed in table s4 and then 5 end - labeled using [ -p]-atp and t4 polynucleotide kinase .
the binding reactions were performed in bandshift buffer ( 10  mm tris ph 7.5 , 1  mm edta , 5% glycerol , 10  mm nacl , 1  mm mgcl2 ) in 20  l reaction mixture containing 0.6  m bldd and radiolabeled dna ( 8,000 c.p.m . ) as well as 0.5  g poly[d(i - c ) ] as nonspecific competitor dna .
when appropriate , increasing amounts of c - di - gmp ( 0.25  1.75  m ) were added to the mixture .
the reaction samples were incubated for 20  min at room temperature followed by electrophoresis on a 5% polyacrylamide gel in 0.5 x tbe ( tris - borate - edta ) buffer at 80v for 105  min .
chromatin immunoprecipitations were performed as previously described ( bush et  al . , 2013 ) , except that an anti - bldd polyclonal antibody was used and pulled down with protein a - sepharose beads . library construction ,
sequencing and chip - seq data analyses were all carried out as previously described ( bush et  al . , 2013 ) .
the his6-bldd - dbd and his6-bldd - ctd proteins were dialyzed into crosslinking buffer ( 100  mm nah2po4 , 150  mm nacl , ph 8) and then incubated at room temperature for 30  min in 20  l reaction samples containing 10  m protein , 1  mm disuccinimidyl suberate ( dss ) in dimethylsulfoxide ( dmso ) , and c - di - gmp as indicated .
the reaction was stopped by adding 50  mm tris ph 8 and incubation for 15  min followed by addition of sds sample buffer and heating to 95c for 10  min .
samples were separated on a 15% sds polyacrylamide gel and visualized by coomassie staining . radiolabeled c - di - gmp was synthesized in  vitro using [ -p]-gtp and the purified diguanylate cyclase pled as described ( paul et  al . , 2004 ) . the dracala assays ( roelofs et  al . , 2011 )
were performed using 2  g of his6-bldd or its n - terminally his - tagged domains that were incubated with 11  nm p - c - di - gmp in dgc buffer ( 250  mm nacl , 25  mm tris ph 8 , 10  mm mgcl2 , 5  mm -mercaptoethanol ) . for competition experiments ,
266  m cold c - di - gmp or gtp were added to the reaction .
after a 5  min incubation at room temperature , 5  l of the binding sample were spotted onto nitrocellulose membrane and the dried membranes were analyzed using a phosphorimager . for structural studies on the ctd ,
the regions encoding residues 80 - 166 ( s.  venezuelae bldd ) and 80 - 167 ( s.  coelicolor bldd ) were cloned into the pet15b vector and the proteins induced at 37c and purified via ni - nta chromatography .
the his - tags were removed from the proteins used for structural studies by thrombin cleavage .
crystals of the s.  venezuelae bldd ctd-(c - di - gmp ) complex , which assumed the trigonal space group , p32 , were obtained using protein at 30  mg / ml and 1  mm c - di - gmp .
crystals were produced via the hanging drop vapor diffusion method and mixing the complex 1:1 with 28% peg 1500 , 100  mm sodium acetate , ph 5.5 .
the s.  venezuelae bldd ctd contains no methionines and hence for phasing , leu92 and ile135 were substituted with methionines .
semet(l92m / i135 m ) bldd ctd was expressed using the methionine inhibitory pathway and the protein purified and crystallized with c - di - gmp as per the wt ctd .
the selenomethionine - substituted l92m / i135 m protein crystallized in the wild - type protein p32 space group .
a second crystal form of the s.  venezuelae bldd ctd-(c - di - gmp ) complex was grown with protein at 20 - 40  mg / ml and 1  mm c - di - gmp using 20% peg 2000 monomethyl ether , 100  mm mes , ph 6.0 , as a crystallization reagent and took the orthorhombic space group p21212 .
the third crystal form of the s.  venezuelae bldd ctd-(c - di - gmp ) complex was produced with protein at 10  mg / ml and 1  mm c - di - gmp using 1.2  m sodium / potassium phosphate , 50  mm citrate ph 5.6 .
crystals were obtained for the s.  coelicolor bldd ctd-(c - di - gmp ) complex using 25  mg / ml protein , 1  mm c - di - gmp and mixing the complex 1:1 with a reservoir comprised of 1.4  m sodium / potassium phosphate , 100  mm hepes ph 7.5 .
multiple wavelength anomalous diffraction ( mad ) data were collected for a semet(l92m / i135 m ) s.  venezuelae bldd ctd-(c - di - gmp ) crystal to 2.28   resolution at als ( advanced light source , berkeley , ca , usa ) beamline 8.3.1 ( table s1 ) .
the data were processed using mosflm and the heavy atom substructure was obtained via solve ( terwilliger and berendzen , 1999 ) .
phenix was used for final phasing and density modification ( adams et  al . , 2010 ) .
the crystal contains 12 protein molecules in the asymmetric unit ( asu ) and each of the six dimers is glued together by four c - di - gmp molecules .
the six c - di - gmp complexed dimers are essentially identical ( figure  4d ) .
final refinement was done using a data set collected to 1.95   resolution for a semet(blddl92 m ) ctd-(c - di - gmp ) crystal .
a wt data set was also collected to 2.2   resolution and the structure was identical to the l92 m and l92m / i135 m structures .
the final 1.95   resolution - structure contains residues 84 - 161 for each of the 12 subunits and 24 c - di - gmp molecules ( table s2 ) .
data were collected to 1.75   , 2.25   and 2.33   resolution for the s.  venezuelae and s.  coelicolor bldd ctd-(c - di - gmp ) c2221 forms and the s.  venezuelae bldd ctd-(c - di - gmp ) p21212 crystal form , respectively , and the structures solved by molecular replacement ( mr ) .
the s.  coelicolor bldd ctd and s.  venezuelae bldd ctd c2221 crystal forms contain a ctd dimer and four c - di - gmp molecules in the asu and the s.  venezuelae bldd c - domain-(c - di - gmp ) p21212 crystal form contains 10 subunits ( five dimers ) , and 20 c - di - gmp molecules .
the structures were solved by mr using the program phaser ( mccoy et  al . , 2007 ) .
the topology of every bldd ctd is 1 ( residues 84 - 88)-1 ( residues 89 - 93)-2 ( residues 98 - 113)-2 ( residues 120 - 124)-3 ( residues 128 - 136)-4 ( residues 140 - 149)-5 ( residues 154 - 160 ) .
the bldd dna binding domain ( bldd dbd ) , encoding residues 1 - 79 , was cloned into pet15b , expressed in e.  coli bl21(de3 ) and the protein purified via ni - nta chromatography .
prior to crystallization , the hexa - his tag was removed via thrombin cleavage and the protein further purified by size exclusion chromatography .
crystals were grown by mixing the protein ( 40  mg / ml ) 1:1 with a reservoir consisting of 35% peg 400 , 0.1  m mgcl2 and 0.1  m tris ph 7.5 .
x - ray intensity data were collected to 2.8   resolution at als beamline 8.3.1 and processed with mosflm .
the rsym and i/(i ) for the data are 11.3% ( 38.4% ) and 11.4 ( 4.3 ) , respectively , where the values in parentheses indicate data from the highest resolution shell .
the structure , which contains 3 subunits in the asu ( 2 subunits form a dimer and crystal symmetry generates a second dimer ) , was solved with phaser using a single s.  coelicolor dbd subunit ( pdb code 2ewt ) as the search model .
the final model contains residues 3 - 71 of each subunit and was refined using phenix to final rwork / rfree values of 23.7%/28.9% , respectively ( adams et  al . , 2010 ) .
crystals of the full - length ( fl ) s.  venezuelae bldd-(c - di - gmp)-21-mer complex were grown by using protein in which the n - terminal hexa - his tag had been cleaved and incubated with 1  mm c - di - gmp ( final concentration ) .
this bldd-(c - di - gmp ) solution was mixed in a 1:1 molar ratio of bldd dimer to 21-mer dna duplex ( 5-cccctcacgctgcgtgacggg-3 , with the canonical bldd box underlined , annealed to its complementary oligodeoxynucleotide ) for crystallization trials .
crystals were grown by mixing the protein - dna complex 1:1 with the crystallization solution composed of 100  mm sodium citrate tribasic / citric acid ph 4.0 and 200  mm ammonium sulfate .
the crystals take the trigonal space group , p3221 , with a  = b  = 114.0   , c  = 95.2   and contain a bldd dimer-(c - di - gmp)-21-mer duplex in the asu .
data were collected to the limiting resolution of 4.5   . the rsym  = 9.0% ( 86.0% ) and i/(i )  = 5.8 ( 1.8 ) , where the values in parentheses are for the highest resolution shell .
first , bldd dna binding domain - dna complex models were constructed based on the sinr - dna ( pdb code 3zkc ) or  repressor - dna ( pdb code 1lmb ) complex structures and used in the mr program phaser ( mccoy et  al . , 2007 ) .
this solution was then used as a static model with the ctd-(c - di - gmp ) dimeric structure as a search model .
, the structure was subjected to rigid body refinement only ( rwork / rfree  = 33.0%/38.9% , respectively ) . to examine the flexibility of the linker region that connects the bldd dbd and ctd ,
specifically , the accessibility of residue glu80 , which is the only acidic residue in the bldd linker region , was determined by the ability of the glu - c protease to cleave after this residue . in these experiments ,
endo - glu - c ( 100  units / ml ) was a diluted 50 fold into samples of 1 ) fl bldd ( 1  mg / ml ) , 2 ) fl bldd-21-mer dna ( 1  mg / ml protein with 100  m dna ) or 3 ) fl bldd-(c - di - gmp)-21-mer ( 1  mg / ml protein with 100  m 21-mer dna and 1  mm c - di - gmp ) .
the proteolysis buffer was 50  mm sodium phosphate , ph 7.5 and the final protein concentration of each sample was 4  mg / ml .
the samples shown in figure  s6d were taken at a time point of 4  hr .
notably , proteolysis of each sample was identical , demonstrating that ligand binding of neither cognate dna nor c - di - gmp by bldd affect the accessibility of the linker region , which remains flexible and unstructured .
size exclusion chromatography experiments were carried out using a hiload 16/600 superdex 75 pg column .
5  mg of either the wild - type bldd ctd or the quadruple mutant were loaded onto a column that had been pre - equilibrated with 150  mm nacl , 5% glycerol , 20  mm tris hcl ph 7.5 and 3  m c - di - gmp .
the samples were run and eluted with the same c - di - gmp containing buffer .
the elution volume was plotted against a standard curve to determine the relative molecular weights of the samples .
the standard curve was determined using cytochrome c ( 12  kda ) , carbonic anhydrase ( 29  kda ) and albumin ( 66  kda ) .
cryo - sem was performed as previously described ( bush et  al . , 2013 ) . when induced in e.  coli bl21(de3 ) at 37c , bldd ctd samples contained little to no c - di - gmp contamination , which was supported by a280/a260 values of the purified protein , which were approximately 1.6 .
however , to ensure that there was no endogenous c - di - gmp present in samples used for binding affinity measurements , the bldd ctd was expressed and purified in sf9 insect cells . for these studies , a gene encoding the same s.  venezuelae bldd ctd region that was expressed in e.  coli was codon optimized for expression in insect cells ( genscript , piscataway , nj , usa ; http://www.genscript.com ) and subcloned into the expression vector f1 , which was transfected into the dh10bac strain for generation of the recombinant bacmid .
rbacmids were then transfected in sf9 insect cells with cellfectin ii and the cells incubated in sf-900 ii sfm for 56  days before harvest .
the supernatant was collected for the p1 viral stock and p2 was amplified for later infection .
sf9 cells expressing bldd ctd were harvested at 72  hr post infection and cells were lysed into 25  mm tris ph 7.5 , 300  mm nacl and 5% glycerol with protease inhibitors .
the protein was purified from the supernatant by ni - nta followed by size exclusion chromatography and was > 90% pure as assessed by sds - page analysis . to measure binding , 2-o-(6-[fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate ( 2-fluo - ahc - c - di - gmp ) ,
this molecule is conjugated via a 9 atom spacer to one of the 2 hydroxyl groups of the c - di - gmp , hence meeting the structural requirement for bldd binding that only one 2 hydroxyl group be unmodified and the other available for the interactions observed in the bldd-(c - di - gmp ) structures ( figure  5 ) .
binding was carried out at 25c in a buffer of 150  mm nacl and 25  mm tris - hcl ph 7.5 , which contained 1  nm 2-fluo - ahc - c - di - gmp .
increasing concentrations of bldd ctd were titrated into the reaction mixture to obtain the binding isotherms .
after each addition of protein the reaction sample was incubated for 30  min to ensure equilibrium had been reached .
c - di - gmp binding to bldd appears to have the characteristics of a high affinity / slow binding ligand .
the resulting data were plotted using kaleidagraph and curves were fitted to deduce binding affinities . to determine the binding stoichiometry of the bldd ctd-(c - di - gmp ) complex , the same fp binding conditions were used but the total concentration of c - di - gmp ( c - di - gmp  + 2-fluo - ahc - c - di - gmp ) was 25  m , 10-fold above the kd of 2.5  m ensuring stoichiometric binding .
close inspection of the binding isotherm revealed the possibility that there are two nearly identical binding events ( likely corresponding to the binding of each intercalated c - di - gmp dimer ) .
the graph of the resulting data shows a linear increase in the observed mps until saturation of the binding sites , after which the line is flat .
importantly , the final inflection occurs at a bldd monomer concentrations of 12  m , which , when divided by the concentrations of c - di - gmp ( 25  m ) , indicates a stoichiometry of two ctd protomers per four c - di - gmps .
as anticipated from a purely chemical complementarity - orientated argument , fp studies carried out in the same buffer also revealed that wild - type bldd ctd does not bind to c - di - amp .
in addition , the bldd dgr - x8-dqdr ctd mutant did not bind the c - di - gmp probe .
n.t . designed , performed , and interpreted experiments , created figures , and wrote the paper . m.a.s . designed , performed , and interpreted experiments , created figures , and wrote the paper .
r.g.b . designed and interpreted experiments and wrote the paper . m.j.b . designed and interpreted experiments and wrote the paper
 summarythe cyclic dinucleotide c - di - gmp is a signaling molecule with diverse functions in cellular physiology . here 
 , we report that c - di - gmp can assemble into a tetramer that mediates the effective dimerization of a transcription factor , bldd , which controls the progression of multicellular differentiation in sporulating actinomycete bacteria . 
 bldd represses expression of  sporulation genes during vegetative growth in a manner that depends on c - di - gmp - mediated dimerization . 
 structural and biochemical analyses show that tetrameric c - di - gmp links two subunits of bldd through their c - terminal domains , which are otherwise separated by 10   and thus can not effect dimerization directly . 
 binding of the c - di - gmp tetramer by bldd is selective and requires a bipartite rxd - x8-rxxd signature . 
 the findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions