h - ns is an abundant global regulator of transcription and a determinant of nucleoid structure in many bacteria ( 1,2 ) .
this protein is primarily a repressor acting both as a selective silencer of specific genes or regions of the chromosome and also more locally in concert with other transcription factors within a regulatory region ( 3 ) .
genes under h - ns control in enteric bacteria are often responsive to abrupt environmental changes .
such genes include those activated by enhanced osmolarity of the growth medium ( exemplified by prov ) and also genes associated with pathogenicity , notably shigella flexneri virf and the pathogenicity - associated islands in uropathogenic bacteria ( 49 ) .
h - ns binding to dna in vitro is facilitated by structural characteristics of dna ( 1 ) but by itself this mechanism may not provide sufficient selectivity for precise silencing .
however , by combining recent findings we derive here an at - rich sequence motif for directing sequence - specific dna binding by h - ns . depending on their context
such motifs can effect local repression or act more globally altering the packaging of dna thereby silencing the packaged genes .
importantly , the proximity of these sites within putative silenced regions indicates how h - ns silencing can be nucleated and propagated over a substantial length of the bacterial chromosome .
we conclude from an analysis of genes containing h - ns binding sites that a major role of h - ns , and also possibly its paralog , is to regulate functional systems as a whole rather than selectively targeting individual regulatory or structural genes .
in addition to structurally determined dna binding ( 1 ) , two recent findings suggest that h - ns binding can be highly sequence specific .
first , two tight binding sites of identical sequence with a kd of 15 nm were identified in the escherichia coli prou operon ( 10 ) .
importantly , this sequence also directed tight h - ns binding when placed in a completely different sequence context , thus excluding any more extensive structure - specific recognition .
second , the h - ns dna binding domain , in isolation , selectively footprinted particular sequences of varying extents within the regulatory regions of several h - ns sensitive genes ( 11 ) . although both results are indicative of sequence - specific binding neither individually is sufficient to identify a  consensus sequence .
however , inspection of the footprinted sequences revealed that they all contained at least one section with high similarity to the high - affinity sites in prov . from the identified sequences a template motif
was derived ( table 1 ) . because the derived motif depended on the assumption that high - affinity binding sites are related in sequence we used the motif to predict three potential h - ns binding sites in the fis promoter from positions 150 to + 1 .
these predicted sites , although imperfect fits to the derived motif , confirmed a footprinting analysis of this promoter ( supplementary data ) and thus provide a validation of the derived sequence template for h - ns binding in vitro ( table 1 ) .
combining the sequences for all identified sites yields the 10 bp motif tcgataaatt ( figure 1 ) .
the base composition , 78% at , is fully consistent with the documented preferred composition of h - ns targets in vivo ( 12 ) .
second , a centrally positioned t - a base step is found in all identified sites . this step would confer thermal instability and both axial and torsional flexibility on the binding sites ( 13 ) . 

table 1.derivation of a dna binding motif template for h - nsgenesitefit(/10)prov 1tcgatatatt9prov 2tcgatatatt9gadacggataaatc7gadxtcgttaaaat9virftagattaagt7.5cspaccgattaatc7.5hnstccttacatt8orictgtatacttt6templatetcgttaaatta tfis 1gcgataatta7fis 2gcatttaaaa5.5fis 3gcgactaaaa5.5the prov sequences are taken from ref .
( 10 ) and the gada , gadx , virf , cspa , hns and oric sequences from ref . ( 11 ) .
figure 1.(a ) logo representation of the experimentally determined h - ns binding motif derived from sequences in table 1 .
( b ) logo representation of the predicted h - ns binding motif derived from identified occurrences in the set of differentially expressed genes responding to h - ns removal under different growth conditions .
( c ) average at content profiles of sequences from 300 to + 100 bp relative to the start of translation for sets of genes differentially expressed in hns cells but containing or lacking a predicted h - ns binding site .
derivation of a dna binding motif template for h - ns the prov sequences are taken from ref .
( 10 ) and the gada , gadx , virf , cspa , hns and oric sequences from ref . ( 11 ) .
( a ) logo representation of the experimentally determined h - ns binding motif derived from sequences in table 1 . ( b ) logo representation of the predicted h - ns binding motif derived from identified occurrences in the set of differentially expressed genes responding to h - ns removal under different growth conditions . ( c )
average at content profiles of sequences from 300 to + 100 bp relative to the start of translation for sets of genes differentially expressed in hns cells but containing or lacking a predicted h - ns binding site .
however , in vivo binding sites have been identified by h - ns - dna chip - on - chip experiments ( 12,1416 ) .
although such experiments do not provide the resolution of in vitro footprinting , they give valuable genome - scale information on locations where h - ns binds dna . we can therefore ask whether sequences related to the binding motif occur at or close to the identified segments .
we found that of the top 99 highest scoring sites identified by grainger et al .
( 16 ) the motif occurred in 59 within 250 bp of the reported signal peak ( supplementary data 1 ) .
as a control , we randomly picked 99 regions of 500 bp in length and asked what fraction contained a high - affinity h - ns binding site .
this calculation was repeated 1000 times and revealed that the average number of expected binding sites was 39 , with a standard deviation of 4.5 binding sites .
therefore , our observation that 59 of the 99 high - confidence regions have a high - affinity h - ns binding site is statistically significant ( z - score = 4.5 ; p - value < 10 ) . we conclude that the h - ns binding motif that we identified in vitro is also characteristic of many in vivo binding sites .
this analysis also revealed that sites identified by the chip - on - chip protocol show a significant enrichment for at .
the average at content of the high - confidence h - ns bound sites was 61.4% .
control experiments where we analyzed at content for a thousand random 500 bp regions from the genome showed that the average expected value was 49.2% .
thus , the observed value of 61.4% represents a difference of > 21 sds from what is expected by chance . in another independent control experiment ,
we first identified 1000 random 500 bp segments that had a predicted binding site and analyzed their at content .
this investigation revealed that the average expected at content for random 500 bp segments that contained a motif was 52% .
we therefore conclude that the sites identified by chip - on - chip are , on average , substantially enriched in at content but randomly selected sequences that may or may not contain a site are not . in the absence of other data ,
the binding of h - ns to a site in vivo , or even the presence of a site , is not necessarily indicative of a biologically relevant role . to assess the functionality of the h - ns binding motif
, we asked whether its occurrence correlated with genes known to be regulated by h - ns in vivo . to test whether such a correlation exists ,
we scanned two sets of genes previously identified by microarray analysis to be derepressed in an e. coli strain lacking h - ns ( 17 ) .
since h - ns is only one component of a complex regulatory system which includes rpos and lrp as well as other dna binding proteins ( 1,18 ) , derepression could be either a direct or an indirect consequence of h - ns removal and therefore not all genes identified by these screens are likely to be direct h - ns targets .
the first set of scanned genes was initially compiled to include genes responding to h - ns removal under different growth conditions .
we found that of the 57 genes scanned 33 ( or 57.9% ) contained one or more sequences related to the h - ns binding motif within a region spanning positions 300 to + 100 bp relative to the first nucleotide of the translation start site ( see methods section ) .
the positive hits included the genes ade , fimd , gada , hlye , ompf , osmc , prov ( supplementary data 2 ) , all of which have previously been independently shown to be regulated by h - ns .
the second set of scanned genes were initially characterized as those which were differentially expressed in isogenic hns+ and hns strains on either reduction or increase of the natural superhelical density ( 17 ) . in this set
, we found that of the 211 genes scanned 98 ( or 46.4% ) registered as positive hits ( supplementary data 3 ) . these included several additional genes , including the hde locus and many genes involved in flagella formation , previously known to be under h - ns control . combining both sets
116 putative target genes were identified , 32 of which contained more than one copy of the motif .
another question of interest was whether the genes containing the h - ns motif differ in base composition from those that do not . since the preferred in vivo targets of h - ns are generally at - rich ( 12 ) , we compared the base composition of these two classes of genes for the region from 300 to + 100 bp relative to the translation start site ( see methods section ) .
we found that for the first set of scanned genes ( expression change in the absence of h - ns ) the at content of the genes containing the h - ns motif was , on average , consistently 10% above the average at content both of the genes apparently lacking the h - ns motif and also the average composition in this region for all e. coli genes ( figure 1 ; p - value < 10 ; wilcoxon signed - rank test ) .
for the second set of scanned genes ( initially assayed under conditions of hyper- and hypo - negative supercoiling ) , the same differential was observed between the genes containing the h - ns motif and the average composition in this region for all e. coli genes ( p - value < 10 ) .
however , for this set the at content of the genes apparently lacking the h - ns motif was intermediate between those containing the h - ns motif and all e. coli genes ( p - value < 10 ) . by including the additional putative h - ns motifs identified in our sequence screen , we refined the logo for h - ns binding ( figure 1 ) and observed little change from the input motif .
again , the most highly conserved feature of the individual identified motifs was a centrally placed t - a base step . in the very few cases where this
was absent it was replaced by c - a , the second most flexible base step ( 13 ) . by their nature
, microarray screens used to identify genes whose expression is hns sensitive would not necessarily reveal all genes under direct h - ns control .
for example , one such well - characterized gene , bgl , remains uninduced under most laboratory conditions ( 19 ) .
we therefore scanned the genes compiled in the regulondb database ( 20 ) as well - characterized targets of h - ns .
a total of 24 out of 39 ( or 61.5% ) contained sequences with a reasonable match to the binding site logo ( figure 2a ) .
we noted that the bgl regulatory region , in addition to containing sequences closely related to the template , also contained several similar sequences that fitted the template less closely . using the criterion that such 
secondary sequences should contain a run of at least six consecutive base - pairs and a t - a base step we identified 10 motifs of this type associated with the bgl regulatory elements ( supplementary data ) .
a similar pattern of multiple primary and secondary sequences was also observed in a number of other genes known to be sensitive to h - ns .
these included eltab , flga / flgb , hdeab , htda , micf , spvr , virf and yghj ( 6,7,2123 ) . 

figure 2.(a ) analysis of known transcriptional regulatory network of h - ns from regulondb for occurrence of the binding motif template .
the arrows indicate that one protein may directly affect the expression of another but do not indicate whether such regulation would be positive or negative .
( a ) analysis of known transcriptional regulatory network of h - ns from regulondb for occurrence of the binding motif template .
the arrows indicate that one protein may directly affect the expression of another but do not indicate whether such regulation would be positive or negative .
although the genes silenced by h - ns appear to contain multiple putative sites for h - ns binding , there are other genes for which h - ns appears to bind to more isolated sites .
one such gene is nir ( 3 ) where h - ns binds to the upstream region in concert with fis and ihf to repress promoter activity .
this region contains two potential h - ns binding sites ( gggtatattg and ttgtataaat ) correlating with the h - ns footprint . both contain the  core of the consensus and the presence of two t - a steps indicates that the sequences are unlikely to confer coherent intrinsic curvature ( 24 ) .
since it is well established that h - ns regulates many genes correlated with enteric pathogenicity of e. coli and salmonella typhimurium ssp .
, we asked whether putative h - ns binding sites could be identified within plasmids such as po157 and r27 that confer pathogenicity and also within the pathogenicity - associated islands in the uropathogenic e. coli strain 536 ( 9 ) . in all cases , we observed a substantial number of positive hits , respectively 19 , 80 and 211 genes ( figure 3 ) . in many cases ,
we note that particularly in the case of the pathogenicity - associated islands of strain 536 there is a strong tendency for the identified genes to be clustered . on average within the islands there
although certain predicted sites can be associated with more than one gene ( e.g. when transcription is divergent ) , the analysis also reveals that there are regions within all of the pathogenicity islands where the spacing between strong sites is 8 genes .
table 2.average number of genes between identified h - ns binding motifs in the pathogenicity - associated islands in e. coli u536islandaverage number of genes between motifspai12.40pai22.40pai32.38pai42.71pai52.95avg2.57 
 figure 3.maps of occurrence of sequences with close matches to the binding motif template in selected regions of ( a ) e. coli k12 , ( b ) uropathogenic e. coli 536 ( c ) plasmids po157 ( top ) and r27 ( bottom ) .
average number of genes between identified h - ns binding motifs in the pathogenicity - associated islands in e. coli u536 maps of occurrence of sequences with close matches to the binding motif template in selected regions of ( a ) e. coli k12 , ( b ) uropathogenic e. coli 536 ( c ) plasmids po157 ( top ) and r27 ( bottom ) .
in addition to structurally determined dna binding ( 1 ) , two recent findings suggest that h - ns binding can be highly sequence specific .
first , two tight binding sites of identical sequence with a kd of 15 nm were identified in the escherichia coli prou operon ( 10 ) .
importantly , this sequence also directed tight h - ns binding when placed in a completely different sequence context , thus excluding any more extensive structure - specific recognition .
second , the h - ns dna binding domain , in isolation , selectively footprinted particular sequences of varying extents within the regulatory regions of several h - ns sensitive genes ( 11 ) . although both results are indicative of sequence - specific binding neither individually is sufficient to identify a  consensus sequence .
however , inspection of the footprinted sequences revealed that they all contained at least one section with high similarity to the high - affinity sites in prov . from the identified sequences a template motif
was derived ( table 1 ) . because the derived motif depended on the assumption that high - affinity binding sites are related in sequence we used the motif to predict three potential h - ns binding sites in the fis promoter from positions 150 to + 1 .
these predicted sites , although imperfect fits to the derived motif , confirmed a footprinting analysis of this promoter ( supplementary data ) and thus provide a validation of the derived sequence template for h - ns binding in vitro ( table 1 ) .
combining the sequences for all identified sites yields the 10 bp motif tcgataaatt ( figure 1 ) .
the base composition , 78% at , is fully consistent with the documented preferred composition of h - ns targets in vivo ( 12 ) .
second , a centrally positioned t - a base step is found in all identified sites . this step would confer thermal instability and both axial and torsional flexibility on the binding sites ( 13 ) . 

table 1.derivation of a dna binding motif template for h - nsgenesitefit(/10)prov 1tcgatatatt9prov 2tcgatatatt9gadacggataaatc7gadxtcgttaaaat9virftagattaagt7.5cspaccgattaatc7.5hnstccttacatt8orictgtatacttt6templatetcgttaaatta tfis 1gcgataatta7fis 2gcatttaaaa5.5fis 3gcgactaaaa5.5the prov sequences are taken from ref .
( 10 ) and the gada , gadx , virf , cspa , hns and oric sequences from ref . ( 11 ) .
figure 1.(a ) logo representation of the experimentally determined h - ns binding motif derived from sequences in table 1 .
( b ) logo representation of the predicted h - ns binding motif derived from identified occurrences in the set of differentially expressed genes responding to h - ns removal under different growth conditions .
( c ) average at content profiles of sequences from 300 to + 100 bp relative to the start of translation for sets of genes differentially expressed in hns cells but containing or lacking a predicted h - ns binding site .
derivation of a dna binding motif template for h - ns the prov sequences are taken from ref .
( 10 ) and the gada , gadx , virf , cspa , hns and oric sequences from ref . ( 11 ) .
( a ) logo representation of the experimentally determined h - ns binding motif derived from sequences in table 1 . ( b ) logo representation of the predicted h - ns binding motif derived from identified occurrences in the set of differentially expressed genes responding to h - ns removal under different growth conditions . ( c )
average at content profiles of sequences from 300 to + 100 bp relative to the start of translation for sets of genes differentially expressed in hns cells but containing or lacking a predicted h - ns binding site .
however , in vivo binding sites have been identified by h - ns - dna chip - on - chip experiments ( 12,1416 ) .
although such experiments do not provide the resolution of in vitro footprinting , they give valuable genome - scale information on locations where h - ns binds dna . we can therefore ask whether sequences related to the binding motif occur at or close to the identified segments .
we found that of the top 99 highest scoring sites identified by grainger et al .
( 16 ) the motif occurred in 59 within 250 bp of the reported signal peak ( supplementary data 1 ) .
as a control , we randomly picked 99 regions of 500 bp in length and asked what fraction contained a high - affinity h - ns binding site .
this calculation was repeated 1000 times and revealed that the average number of expected binding sites was 39 , with a standard deviation of 4.5 binding sites .
therefore , our observation that 59 of the 99 high - confidence regions have a high - affinity h - ns binding site is statistically significant ( z - score = 4.5 ; p - value < 10 ) . we conclude that the h - ns binding motif that we identified in vitro is also characteristic of many in vivo binding sites .
this analysis also revealed that sites identified by the chip - on - chip protocol show a significant enrichment for at .
the average at content of the high - confidence h - ns bound sites was 61.4% .
control experiments where we analyzed at content for a thousand random 500 bp regions from the genome showed that the average expected value was 49.2% .
thus , the observed value of 61.4% represents a difference of > 21 sds from what is expected by chance . in another independent control experiment ,
we first identified 1000 random 500 bp segments that had a predicted binding site and analyzed their at content .
this investigation revealed that the average expected at content for random 500 bp segments that contained a motif was 52% .
we therefore conclude that the sites identified by chip - on - chip are , on average , substantially enriched in at content but randomly selected sequences that may or may not contain a site are not .
in the absence of other data , the binding of h - ns to a site in vivo , or even the presence of a site , is not necessarily indicative of a biologically relevant role . to assess the functionality of the h - ns binding motif
, we asked whether its occurrence correlated with genes known to be regulated by h - ns in vivo .
to test whether such a correlation exists , we scanned two sets of genes previously identified by microarray analysis to be derepressed in an e. coli strain lacking h - ns ( 17 ) .
since h - ns is only one component of a complex regulatory system which includes rpos and lrp as well as other dna binding proteins ( 1,18 ) , derepression could be either a direct or an indirect consequence of h - ns removal and therefore not all genes identified by these screens are likely to be direct h - ns targets .
the first set of scanned genes was initially compiled to include genes responding to h - ns removal under different growth conditions .
we found that of the 57 genes scanned 33 ( or 57.9% ) contained one or more sequences related to the h - ns binding motif within a region spanning positions 300 to + 100 bp relative to the first nucleotide of the translation start site ( see methods section ) .
the positive hits included the genes ade , fimd , gada , hlye , ompf , osmc , prov ( supplementary data 2 ) , all of which have previously been independently shown to be regulated by h - ns .
the second set of scanned genes were initially characterized as those which were differentially expressed in isogenic hns+ and hns strains on either reduction or increase of the natural superhelical density ( 17 ) . in this set
, we found that of the 211 genes scanned 98 ( or 46.4% ) registered as positive hits ( supplementary data 3 ) .
these included several additional genes , including the hde locus and many genes involved in flagella formation , previously known to be under h - ns control . combining both sets
116 putative target genes were identified , 32 of which contained more than one copy of the motif .
another question of interest was whether the genes containing the h - ns motif differ in base composition from those that do not .
since the preferred in vivo targets of h - ns are generally at - rich ( 12 ) , we compared the base composition of these two classes of genes for the region from 300 to + 100 bp relative to the translation start site ( see methods section ) .
we found that for the first set of scanned genes ( expression change in the absence of h - ns ) the at content of the genes containing the h - ns motif was , on average , consistently 10% above the average at content both of the genes apparently lacking the h - ns motif and also the average composition in this region for all e. coli genes ( figure 1 ; p - value < 10 ; wilcoxon signed - rank test ) .
for the second set of scanned genes ( initially assayed under conditions of hyper- and hypo - negative supercoiling ) , the same differential was observed between the genes containing the h - ns motif and the average composition in this region for all e. coli genes ( p - value < 10 ) .
however , for this set the at content of the genes apparently lacking the h - ns motif was intermediate between those containing the h - ns motif and all e. coli genes ( p - value < 10 ) . by including the additional putative h - ns motifs identified in our sequence screen , we refined the logo for h - ns binding ( figure 1 ) and observed little change from the input motif .
again , the most highly conserved feature of the individual identified motifs was a centrally placed t - a base step . in the very few cases where this was absent
it was replaced by c - a , the second most flexible base step ( 13 ) . by their nature
, microarray screens used to identify genes whose expression is hns sensitive would not necessarily reveal all genes under direct h - ns control .
for example , one such well - characterized gene , bgl , remains uninduced under most laboratory conditions ( 19 ) .
we therefore scanned the genes compiled in the regulondb database ( 20 ) as well - characterized targets of h - ns .
a total of 24 out of 39 ( or 61.5% ) contained sequences with a reasonable match to the binding site logo ( figure 2a ) .
we noted that the bgl regulatory region , in addition to containing sequences closely related to the template , also contained several similar sequences that fitted the template less closely . using the criterion that such 
secondary sequences should contain a run of at least six consecutive base - pairs and a t - a base step we identified 10 motifs of this type associated with the bgl regulatory elements ( supplementary data ) .
a similar pattern of multiple primary and secondary sequences was also observed in a number of other genes known to be sensitive to h - ns . these included eltab , flga / flgb , hdeab , htda , micf , spvr , virf and yghj ( 6,7,2123 ) . 

figure 2.(a ) analysis of known transcriptional regulatory network of h - ns from regulondb for occurrence of the binding motif template .
the arrows indicate that one protein may directly affect the expression of another but do not indicate whether such regulation would be positive or negative .
( a ) analysis of known transcriptional regulatory network of h - ns from regulondb for occurrence of the binding motif template . genes containing a predicted strong h - ns binding site are underlined .
the arrows indicate that one protein may directly affect the expression of another but do not indicate whether such regulation would be positive or negative .
although the genes silenced by h - ns appear to contain multiple putative sites for h - ns binding , there are other genes for which h - ns appears to bind to more isolated sites .
one such gene is nir ( 3 ) where h - ns binds to the upstream region in concert with fis and ihf to repress promoter activity .
this region contains two potential h - ns binding sites ( gggtatattg and ttgtataaat ) correlating with the h - ns footprint . both contain the  core of the consensus and the presence of two t - a steps indicates that the sequences are unlikely to confer coherent intrinsic curvature ( 24 ) . since it is well established that h - ns regulates many genes correlated with enteric pathogenicity of e. coli and salmonella typhimurium ssp .
, we asked whether putative h - ns binding sites could be identified within plasmids such as po157 and r27 that confer pathogenicity and also within the pathogenicity - associated islands in the uropathogenic e. coli strain 536 ( 9 ) . in all cases
, we observed a substantial number of positive hits , respectively 19 , 80 and 211 genes ( figure 3 ) .
we note that particularly in the case of the pathogenicity - associated islands of strain 536 there is a strong tendency for the identified genes to be clustered . on average within the islands there
although certain predicted sites can be associated with more than one gene ( e.g. when transcription is divergent ) , the analysis also reveals that there are regions within all of the pathogenicity islands where the spacing between strong sites is 8 genes .
table 2.average number of genes between identified h - ns binding motifs in the pathogenicity - associated islands in e. coli u536islandaverage number of genes between motifspai12.40pai22.40pai32.38pai42.71pai52.95avg2.57 
 figure 3.maps of occurrence of sequences with close matches to the binding motif template in selected regions of ( a ) e. coli k12 , ( b ) uropathogenic e. coli 536 ( c ) plasmids po157 ( top ) and r27 ( bottom ) .
average number of genes between identified h - ns binding motifs in the pathogenicity - associated islands in e. coli u536 maps of occurrence of sequences with close matches to the binding motif template in selected regions of ( a ) e. coli k12 , ( b ) uropathogenic e. coli 536 ( c ) plasmids po157 ( top ) and r27 ( bottom ) .
by combining dna footprinting data from different sources , we have identified a dna sequence that correlates with h - ns binding at specific locations in the e. coli genome .
this sequence , or sequences closely related to it , is found in the regulatory regions of genes that are silenced by h - ns ( e.g. bgl and prou ) and also those ( e.g. nir ) in which h - ns acts in cooperation with other abundant dna - binding proteins . in the former class , but not in the latter , abundant at - rich sequences which fit less well to the consensus are interdigitated with the putative high - affinity binding sites .
we suggest that this organization facilitates the  spreading of bound h - ns around nucleating high - affinity sites and the consequent formation of a repressive higher - order structure that is responsible for silencing .
we have validated the identified binding site by three procedures  by showing that it occurs with high frequency in vivo at h - ns binding sites identified by a chip - on - chip protocol , by a search for its occurrence in genes shown to be differentially regulated in hns mutant strains and by direct comparison to reported characterized footprints .
we have further shown that sequences closely related to the consensus are associated with genes implicated in pathogenicity .
our search was restricted to a region spanning 300 to + 100 bp relative to the initiating atg of translated genes and would miss sites located outside this region . indeed , one of the high - affinity binding sites identified by bouffartigues et al .
( 10 ) is located at + 130 bp downstream of the translation start site .
nevertheless , its identification within all the characterized regulatory regions analyzed ( e.g. bgl , prov ) implies that a high - affinity binding site or sites are characteristic of such regions .
our screen also identified putative binding sites in adjacent genes ( e.g. csgd and csge , fima and fimi ) , and genes separated by one other gene within the same operon ( e.g. flgc , flge , flgg ) .
the pattern of sites associated with adjacent genes was particularly apparent both in the r27 plasmid and the pathogenicity - associated islands of e. coli u536 .
our results are consistent with a silencing mechanism in which h - ns binds first to nucleating high - affinity sites ( 10,25 ) , corresponding in principle to the logo identified here , and then polymerizes resulting eventually in the formation of a supercoiled interwound filament containing two dna duplexes joined by protein bridges , concomitantly constraining a dna loop ( 21,2629 ) ( figure 4 ) .
this mode of binding also explains previous observations showing that h - ns binds preferentially to dna containing intrinsically curved regions since the dna in plectonemes is bent both within the interwindings and at the apical loops .
the frequent location of nucleating sites associated with adjacent genes and also their repetition within operons would allow the filament to silence an extensive region .
such a mechanism would be consistent with the silencing of pathogenicity - associated islands in enteropathogenic proteobacteria ( 9 ) .
similarly , the at - rich composition of regions adjacent to the sites we have identified could facilitate spreading of h - ns by interspersing high - affinity nucleating sites with lower affinity at - rich secondary sites . in support of this notion , in the po157 plasmid
the higher at content of regions containing h - ns binding sites relative to those that do not , be a simple consequence of the composition of the site itself since there are extensive regions enriched in at close to the initiation codon that lack identified motifs ( supplementary data 5 ) .
in addition , we have shown that the presence of a motif per se is not necessarily correlated with substantial at enrichment . at -
enriched regions are thus extensive and may contain zero to several examples of the motif .
we suggest that nucleation of dna binding of a structural component of a protein  dna array , such as an h - ns - dna fiber , followed by spreading , may constitute a general mechanism for organizing chromatin both in bacteria and eukaryotes . 

initial nucleation of h - ns binding at high - affinity sites is following by spreading and condensation of h - ns on supercoiled dna to generate a silenced fiber .
blue squares represent high - affinity h - ns binding sequences ( nucleating sites ) and green and yellow circles represent bound h - ns on nominal distal and proximal sides , respectively , of a promoter .
initial nucleation of h - ns binding at high - affinity sites is following by spreading and condensation of h - ns on supercoiled dna to generate a silenced fiber .
blue squares represent high - affinity h - ns binding sequences ( nucleating sites ) and green and yellow circles represent bound h - ns on nominal distal and proximal sides , respectively , of a promoter .
the ability of h - ns both to bind locally and to silence longer regions of dna is similar to the dna - binding properties of another abundant global regulator , fis .
both can bind with high specificity to individual sites and can also bind cooperatively to more extensive regions .
these properties allow them to act both as transcription factors and as determinants of nucleoid structure .
another feature of the occurrence of h - ns binding sites is that many genes within a particular regulatory system appear to be targeted .
the h - ns binding motif is associated with not only the genes encoding the structural genes of flagella but also with the specific sigma factor ( flia ) and anti - sigma factor ( flgm ) genes for regulating flagella biosynthesis .
in addition the h - ns binding motif is associated with papx , a gene encoded by the po157 plasmid that is a functional homolog of proteus mirabilis mrpj ( 30 ) and represses the flagellar regulon . in this example , the pattern is indicative of a primary global regulator that controls both positive and negative secondary regulators as well as their targets . to assess the biological relevance of this pattern , we compared the occurrence of the motif with the in vivo binding of h - ns to flagellar genes .
peaks of binding detected by chip - on - chip are located near flgn , flia , flic ( two separate ones ) , flid , flif and fliz ( 14 ) .
nearly all of these , with the exception of the second flic peak and flid , contain the h - ns binding motif .
we conclude that there is a good correlation between the presence of the motif and in vivo binding for this set of genes and that this correlation is indicative of functionality
. however , the effects of hns on the expression of the flagellar regulon are complex . in hns mutants ,
nevertheless , the flhdc master operon is repressed in vitro by h - ns and expression from its promoter is increased in an hns mutant ( 32 ) .
similarly an activator , hdfr , of the master operon , is also negatively regulated by h - ns in vivo ( 33 ) and its gene is associated with a putative h - ns binding site .
these observations show that h - ns can repress certain individual transcriptons within the flagellar regulon , but the regulatory complexity is such that the outcome of h - ns removal is likely only predictable in the context of the complete control network .
we suggest that h - ns acts as a system regulator that is involved in coordinating the expression of the flagellar regulon .
another possible example of this mode of control by h - ns is the mxi - spa type iii secretion regulon ( 34 ) . in this system ,
the activator virb displaces h - ns from repressed promoter sites but does not itself directly affect rna polymerase function ( 35 ) .
an additional aspect of regulation by h - ns is the interdependent regulation of h - ns and its paralogs .
one of the genes identified in our screen was sfh , a paralog of h - ns encoded by the r27 virulence plasmid ( 21,36 ) .
the paralog has the same dna binding domain as h - ns and so is assumed to bind to the same dna sites .
notably the regulatory regions of both the hns gene itself and that of its paralog stpa contain multiple sites with similarities to the h - ns binding site , although in neither case are there sites which correspond to the canonical sequence ( supplementary data 12 ) .
we suggest that interactions between these different , but functionally related proteins serve to act homeostatically to maintain an appropriate overall total concentration of repressors in the cell .
concomitantly , the interactions of h - ns paralog and fis with the regulatory regions of other genes encoding nucleoid - associated proteins ( crp , hupa , hupb ) would similarly act homeostatically to maintain nucleoid structure ( figure 2b ) .
the complete genome , predicted proteome and coding sequence locations for the e. coli strain k12 and the plasmids were obtained from ncbi genbank . the regions from 300 to + 100 bp relative to the translation start site
were scanned for the presence of the binding motif using several publicly available programs such as mast , motifscanner and scanace .
the program weblogo was used to automatically generate logos of the predicted binding sites . for the at content analysis
, the genes were categorized into three classes : ( i ) those that were differentially expressed and predicted to have an h - ns binding site ( direct targets ) , ( ii ) those that were differentially expressed but did not contain a predicted h - ns binding site ( indirect targets ) and ( iii ) those that were not differentially expressed ( control set ) . in order to calculate the mean at content for each position within the sequences of each set
. a window of 250 to + 250 bp relative to the signal center was analyzed for presence of the binding site motif .
 the global transcriptional regulator h - ns selectively silences bacterial genes associated with pathogenicity and responses to environmental insults . 
 although there is ample evidence that h - ns binds preferentially to dna containing curved regions , we show here that a major basis for this selectivity is the presence of a conserved sequence motif in h - ns target transcriptons . 
 we further show that there is a strong tendency for the h - ns binding sites to be clustered , both within operons and in genes contained in the pathogenicity - associated islands . in accordance with previously published findings , we show that these motifs occur in at - rich regions of dna . on the basis of these observations 
 , we propose that h - ns silences extensive regions of the bacterial chromosome by binding first to nucleating high - affinity sites and then spreading along at - rich dna . 
 this spreading would be reinforced by the frequent occurrence of the motif in such regions . 
 our findings suggest that such an organization enables the silencing of extensive regions of the genetic material , thereby providing a coherent framework that unifies studies on the h - ns protein and a concrete molecular basis for the genetic control of h - ns transcriptional silencing . 
INTRODUCTION
RESULTS
Identification of an H-NS DNA binding motif
None
DISCUSSION
MATERIALS AND METHODS
SUPPLEMENTARY DATA