Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding
[['Transcription initiation , the first step in gene expression , is highly regulated to ensure correct timing of developmental processes and the response to environmental stimuli .', 'In bacteria , transcription initiation involves association of RNA polymerase ( RNAP ) with promoter DNA .', 'Core RNAP must associate with a Sigma ( σ ) factor to make sequence-specific contacts with promoter DNA [1] .', 'Following promoter escape , σ factors are released from the elongating RNAP [2] .', 'Bacterial cells often express a single “primary” σ factor and multiple “alternative” σ factors .', 'The primary σ factor is constitutively active and is responsible for transcription of most genes .', 'Alternative σ factors are typically expressed or activated under specific growth conditions and recognize promoters with nucleotide sequences distinct from those recognized by the primary σ factor [3] .', 'Consequently , alternative σ factors govern the transcription of different sets of genes ( regulons ) .', 'Depending on the growth phase , environmental conditions , and developmental stage experienced by the cell , the composition of the pool of active σ factors can vary , allowing for dynamic and rapid expression of different regulons as needed .', 'Escherichia coli has one primary σ factor ( σ70 ) and six alternative σ factors ( σ19 , σ24 , σ28 , σ32 , σ38 and σ54 ) [3] .', 'There are two families of σ factor in bacteria: the σ70 family and the σ54 family .', 'σ54 proteins differ dramatically from those in the σ70 family , both in sequence and domain structure .', 'σ54 promoter elements consist of conserved nucleotides located at -12 and -24 with respect to the transcription start site [4] .', 'This contrasts with members of the σ70 family , which recognize conserved promoter elements located at roughly -10 and -35 with respect to the transcription start site [3] .', 'Unlike the members of the σ70 family , σ54 proteins have been shown to bind promoter DNA independent of core RNAP in vitro [5] .', 'Another distinguishing characteristic of σ54 proteins is their absolute requirement for activator proteins , known as bacterial enhancer binding proteins ( bEBPs ) , to initiate transcription [4 , 6] .', 'bEBPs act in a manner distinct from typical σ70 transcriptional activator proteins: rather than helping to recruit RNAP , like most activators of σ70 , bEBPs use ATP hydrolysis to drive isomerization of RNAP already bound at the promoter [4] .', 'Thus , both active and inactive forms of RNAP:σ54 are bound at promoters .', 'The archetypal member of the σ54 family is σ54 from E . coli .', 'Originally identified as a regulator of genes involved in nitrogen metabolism and assimilation under nitrogen limiting conditions [7] , E . coli σ54 has since been shown to play important regulatory roles in a variety of other cellular processes .', 'Similarly , σ54 homologues in other species regulate a wide range of processes , including flagellar synthesis and virulence [8] .', 'E . coli , and most other bacterial species , have only one σ54 family protein [9] .', 'Genome-wide DNA binding studies have been performed for a handful of σ factors in different bacteria .', 'σ factors show a wide range in the number of sites bound , from just a few sites for extracytoplasmic function ( ECF ) σ70 family factors [10 , 11] to more than a thousand sites for E . coli σ70 [12 , 13] .', 'These studies have revealed new promoters and often help to clarify gene expression changes that are a direct result of proximal RNAP:σ binding .', 'Unexpectedly , some σ factors have been shown to bind extensively inside genes [14 , 15 , 16 , 17 , 18] .', 'Notably , 58% of the σ54 binding sites described in Salmonella Typhimurium occur inside genes [14] .', 'Interestingly , in Vibrio cholerae , only 10% of the described σ54 binding sites occur inside genes [19] , suggesting variation in the regulatory capacity of σ54 across bacterial species .', 'The function , if any , of σ54 binding events inside genes is unknown , with the exception of one promoter that has been shown to drive transcription of an mRNA for the downstream gene , with the RNA having an unusually long 5ʹ UTR [20] .', 'In this study we use chromatin immunoprecipitation ( ChIP ) followed by deep sequencing ( ChIP-seq ) to determine the genome-wide binding profile of σ54 in E . coli .', 'We confirm all but one previously reported promoter and identify 116 novel , high confidence σ54 binding sites .', 'Notably , two thirds of the σ54 binding sites occur inside genes .', 'These intragenic sites are oriented non-randomly , with the majority oriented in the same direction as the overlapping gene .', 'We show that three intragenic binding sites are functional promoters , and conservation analysis suggests that many others are functional under different growth conditions .'], ['We predicted that all σ54-transcribed promoters would be bound by RNAP:σ54 under all growth conditions in which σ54 is expressed .', 'Therefore , we mapped the binding of σ54 using ChIP-seq for cells grown to mid-logarithmic phase in M9 minimal medium .', 'Using a high stringency analysis , we identified 145 ChIP-seq peaks that correspond to putative sites of σ54 binding ( Tables 1 and 2 and S1 Table ) .', 'The identified peaks have a bimodal shape , typical of ChIP-seq data ( Fig 1A ) [21] .', 'We used MEME [22] to identify enriched sequence motifs in the 150 bp regions surrounding each of the putative σ54 binding sites .', 'A motif closely resembling the known σ54–24/-12 promoter elements [6] was identified in 135 of the regions ( Fig 1B ) .', 'This represents a highly significant enrichment for the motif within these sequences ( MEME E-value 1 . 8e-213 ) .', 'Furthermore , the distribution of motif positions within the ChIP-based query sequences was non-random: motifs were far more likely to be located at the center of the query sequence than expected by chance ( p = 1 . 1e-61; Fig 1C ) , as would be expected for genuine σ54 binding sites .', 'ChIP-seq peaks without an associated motif are listed in S1 Table .', 'We selected 13 putative σ54 binding sites for validation .', 'All of these sites are associated with a motif identified by MEME .', 'We used quantitative PCR to measure enrichment of these sites by ChIP ( ChIP-qPCR ) of σ54 in wild type cells or cells in which rpoN is deleted .', 'In all cases , we detected robust enrichment in wild type cells that was significantly higher than that in ΔrpoN cells ( Fig 2A ) .', 'Moreover , ChIP-qPCR enrichment scores correlated well with enrichment measured by ChIP-seq ( R2 = 0 . 71; Fig 2B ) .', 'We also selected five of the ten regions for which we detected a ChIP-seq peak but no motif .', 'We suspected that these were false positives that are commonly found in ChIP-seq datasets for regions that are highly transcribed [12 , 23 , 24 , 25] .', 'We used ChIP-qPCR of σ54 in wild type cells and ΔrpoN cells .', 'We observed no significant difference in enrichment levels between the wild type and ΔrpoN cells ( S1 Fig ) , consistent with these sites being false positives .', 'We conclude that nearly all of the binding sites identified by ChIP-seq , for which we also detected a motif , represent genuine sites of σ54 binding .', 'For all further analyses , we required that the ChIP-enriched sequences include a σ54 promoter motif identified by MEME to be called as a genuine σ54 binding site ( Tables 1 and 2 ) .', 'We refer to these 135 binding sites as “high stringency sites” .', 'Note that we use the term “binding site” rather than “promoter” because we do not know which sites represent functional promoters , as opposed to σ54 binding sites that never lead to productive transcription .', '135 σ54 binding sites represents a very large increase over previously described , experimentally confirmed σ54 promoters , of which there are only 20 [26] .', 'Nonetheless , we suspected that our stringent peak-calling algorithm may have missed some genuine σ54 promoters .', 'Consistent with this , we failed to call a peak upstream of crl , despite two independent reports of σ54 binding [27 , 28] .', 'Visual analysis of the ChIP-seq data for the region upstream of crl indicated a small peak , below the threshold used for peak calling ( S2 Fig ) .', 'We reduced the stringency of our peak-calling algorithm such that it identified an additional 204 “low stringency” peaks ( i . e . 349 total peaks ) , including the site upstream of crl .', 'We then analyzed the sequences surrounding these low stringency peaks using MEME [22] .', 'We identified a highly enriched motif that closely resembles the known σ54–24/-12 promoter elements ( S3A Fig ) .', 'This motif is present in 149 of the 204 sequences ( S2 Table ) and is centrally enriched ( p = 9 . 6e-41; S3B Fig ) .', 'This is a smaller fraction than for the 135 sites identified using the stringent cut-off , indicative of a higher false positive rate .', 'Nevertheless , it suggests that σ54 binds more than 250 sites in the E . coli genome .', 'A recent study using ChIP-chip to map the binding of σ54 across the E . coli genome identified 161 putative σ54 binding sites [29] .', 'We compared putative σ54 binding sites identified in the ChIP-chip study to the 135 high stringency σ54 binding sites we identified , in addition to the 10 sites we identified by ChIP-seq but which lacked a detectable motif .', 'Strikingly , only 67 putative σ54 binding sites overlapped with our own list ( S3 Table ) .', 'We searched for enriched sequence motifs in the remaining 94 putative sites that were unique to the ChIP-chip study .', 'The three most significantly enriched motifs identified by MEME [22] are all repetitive sequences from insertion elements ( S4A Fig ) and are not centrally enriched .', 'There were no significantly enriched motifs that resembled the known σ54 promoter elements [6] .', 'We also used MEME to search for enriched sequence motifs in the putative σ54 binding sites that were unique to our high stringency list .', 'MEME detected a highly enriched motif that resembles the known σ54 promoter elements ( MEME E-value = 5 . 7e-83; S4B Fig ) [6] .', 'This motif is centrally enriched ( p = 4 . 8e-28; S4C Fig ) .', 'We conclude that the majority of the σ54 binding sites identified using ChIP-chip are false positives and that many genuine sites were missed .', 'We compared our high stringency list of 135 σ54 binding sites to those identified in other studies .', 'The EcoCyc database [26] lists 20 σ54 promoters that have been experimentally confirmed .', 'We identified 18 of these ( Table 1 ) .', 'Thus , our approach validates almost all known σ54 promoters .', 'For one of the “known” σ54 promoters that we failed to identify , that for ibpB , we detected background levels of σ54 ChIP signal , suggesting that this is not a genuine σ54 promoter and is misannotated .', 'The EcoCyc database [26] also lists 76 predicted σ54 promoters that have not been experimentally confirmed .', 'These promoters have been predicted based on either DNA sequence or expression microarray data .', 'We confirmed 15 of these predicted sites ( Table 1 ) .', 'A previous study used microarrays to compare expression of cells lacking σ54 to cells transiently overexpressing σ54 [28] .', 'They identified 22 putative , novel σ54 promoters with high confidence .', 'We identified appropriately orientated ChIP-seq peaks ≤500 bp upstream of only five of these genes ( norV , xdhA , ygfK , rutA and ddpX ) .', 'We used MEME [22] to search for enriched motifs in the regions upstream of the 22 putative , novel σ54-transcribed genes identified by [28] ( including the five confirmed by our data ) .', 'There were no significantly enriched motifs that resembled the known σ54 promoter elements ( S5 Fig ) [6] .', 'We conclude that the majority of the σ54 binding sites identified using microarray analysis of RNA levels are false positives and that many genuine sites were missed .', 'We selected all genes for which there was a σ54 binding site positioned <300 bp upstream in the same orientation as the gene .', 'We then searched for enriched Gene Ontology ( GO ) terms using FuncAssociate [30] .', 'We failed to identify any significantly enriched GO terms ( p < 0 . 05 with multiple hypothesis testing correction ) , indicating that σ54-transcribed genes are not , as a group , strongly associated with any specific functions .', 'This is consistent with previous studies that have identified a wide variety of functions for σ54-transcribed genes [6] .', 'With one exception [20] , all previously described , experimentally confirmed σ54 binding sites in E . coli are located in intergenic regions .', 'The distribution with respect to gene position of the 135 high stringency σ54 binding site locations we identified is shown in Fig 3A .', 'As expected , σ54 binding sites as a group are closer to annotated gene starts than random genomic positions ( Fig 3B ) .', 'Surprisingly , 85 of the 135 σ54 binding sites ( 62% ) are located inside genes ( Fig 3A ) .', 'Furthermore , of the 50 σ54 binding sites in intergenic regions , 8 are oriented away from the neighboring gene ( s ) , indicating that they are not promoters for the immediately adjacent genes .', 'Further analysis of the σ54 binding sites inside genes showed that 58 ( 68% ) of these sites are positioned in the sense orientation with respect to the overlapping gene ( Fig 3A ) .', 'This is far more than expected by chance ( Binomial test p = 5e-4 , assuming a 50% random chance of sense/antisense orientation ) .', 'We observed a similar , significant bias ( 79/129; 61% ) for the low stringency sites ( Binomial test p = 0 . 007; S2 Table ) .', 'The distance of intragenic σ54 binding sites from the nearest appropriately oriented gene start is also non-random .', 'Specifically , there are many more sites between 360 and 760 bp upstream of an available gene start than expected by chance ( 36 . 5% of all intragenic sites as compared to 14 . 3% of all intragenic coordinates genome-wide; Fig 3B ) .', 'σ54 can bind promoter DNA in the absence of core RNAP in vitro , although the affinity is lower than that in the context of RNAP holoenzyme [5] .', 'To determine whether σ54 binding in vivo is in the context of RNAP holoenzyme , we used ChIP-seq to map the genome-wide distribution of the β subunit of core RNAP under the same growth conditions used for σ54 ChIP-seq .', 'As shown in Fig 1A , we detected increased local signal for β at sites of σ54 binding .', 'In some cases , especially for intragenic σ54 binding sites , the σ54 peak is located within a transcribed region .', 'Hence , it is more difficult to ascertain σ54-dependent RNAP binding at these positions .', 'Therefore , we determined the median occupancy of RNAP at each of the positions in the 1 kbp region surrounding all σ54 binding sites ( Fig 4; note that each σ54 peak contributes equally in this analysis , regardless of the ChIP-seq signal for σ54 ) .', 'RNAP binding is substantially greater at the exact site of σ54 binding than in the flanking sequence .', 'Furthermore , RNAP binding at the motif center is enriched compared to RNAP binding at randomly selected positions throughout the genome ( Fig 4 ) .', 'We conclude that most or all σ54 binding in vivo is in the context of RNAP holoenzyme .', 'We also noted that the distribution of RNAP binding at σ54 binding sites is symmetric ( Figs 1A and 4 ) , indicative of RNAP that is not actively transcribing RNA .', 'Thus , our data strongly suggest that most or all RNAP:σ54 is transcriptionally inactive under the conditions tested .', 'Furthermore , as described in more detail below , little RNA initiates from identified σ54 binding sites under the conditions used in our study ( Fig 5 and S4 Table ) .', 'It is intriguing that', '( i ) a large proportion of σ54 binding sites is intragenic ( Fig 3A ) ,', '( ii ) 52% of σ54 binding sites are >500 bp from an annotated gene start ( Fig 3B ) ,', '( iii ) σ54 binding at most sites appears to be transcriptionally inactive ( Fig 4 ) , and', '( iv ) σ54 has been previously shown to repress transcription when its binding site overlaps another promoter [27 , 31] .', 'Based on these observations , we postulated that intragenic σ54 binding may reduce expression of the overlapping genes by acting as a transcriptional “roadblock” [32] .', 'To test this hypothesis , we compared the expression of all genes using RNA-seq in cells lacking σ54 and cells transiently overexpressing σ54 .', 'We observed significant ( ≥ 2-fold regulation with an estimated False Discovery Rate ≤ 0 . 01 ) expression differences for 465 genes .', '272 genes were up-regulated following σ54 overexpression , while 193 were down-regulated ( Fig 5 and S4 Table ) .', 'Of the 85 genes with intragenic σ54 binding sites , only ybaT , erfK and clpB were significantly down-regulated ( S4 Table ) .', 'Down-regulation of three out of 85 genes is not significantly more than expected by chance given the total number of down-regulated genes ( Fisher’s exact test p = 0 . 47 ) .', 'Therefore , it is unlikely that RNAP:σ54 acts as a transcriptional roadblock under these conditions .', 'We also examined the genes that were up-regulated upon σ54 overexpression .', 'Only 5 genes with intergenic σ54 binding sites upstream were significantly up-regulated .', 'These genes are all known to be transcribed by RNAP:σ54 .', 'For each gene , regulation by a known bEBP has been described previously: glnA and glnH by NtrC [33] , pspA and pspG by PspF [34] and glmY by GlrR ( Fig 5 ) [35] .', 'No other known RNAP:σ54-transcribed genes were significantly up-regulated .', 'Ten genes with intragenic σ54 sites were significantly up-regulated following σ54 overexpression .', 'Up-regulated genes carB , purK , wbbK , yhbE and rplD contain a σ54 motif in the sense orientation relative to the overlapping gene .', 'However , genes immediately upstream were also up-regulated , suggesting an indirect effect of σ54 overexpression on whole operons .', 'rpmJ , metQ , yajG and cysK were also up-regulated by σ54 overexpression .', 'However , the motif associated with these binding sites is in the antisense orientation .', 'Finally , metF , a gene up-regulated upon σ54 overexpression , contains an intragenic σ54 binding site in the sense orientation that is not associated with up-regulation of an upstream gene .', 'However , the σ54 site is located near the 3ʹ-end of the gene , suggesting that it is not responsible for the observed increase in expression .', 'We conclude that up-regulation of genes that contain intragenic σ54 binding sites is not due to binding of σ54 at these positions , but rather to indirect effects of σ54 overexpression .', 'Our RNA-seq analysis indicates that only three bEBP activators , NtrC , PspF and GlrR , are active under the conditions used , most likely at a very low level ( Fig 5 and S4 Table ) .', 'A single condition where most bEBPs are induced has not been determined; hence , most σ54 promoters will be inactive under a given condition .', 'The bEBP NtrC is highly active in nitrogen-limiting conditions [20] .', 'We compared our ChIP-seq data to published microarray data from E . coli grown under nitrogen-rich conditions ( NtrC inactive ) , and conditions known to induce activity of NtrC [36] .', 'Not all of the σ54 initiated genes are expected to be up-regulated under these conditions , just those under control of NtrC .', 'There are 15 previously described NtrC-regulated operons in E . coli [20 , 37] .', 'All of these operons are associated with a σ54 ChIP-seq peak ( our study ) , and most are associated with observed increases in expression in nitrogen-limiting conditions , e . g . nac ( Fig 6A ) [29] .', 'We observed two additional intergenic ChIP-seq peaks associated with increased expression of the downstream genes , hypA and zraP , in nitrogen-limiting conditions ( Fig 6A ) .', 'hypA and zraP are both known to be transcribed by RNAP:σ54; however , transcription of these genes is activated by bEBPs other than NtrC .', 'Specifically , transcription of hypA is activated by FhlA in response to anaerobiosis and the presence of formate [38] , and transcription of zraP is activated by ZraR in response to high zinc concentrations [39] .', 'Thus , our data suggest either that hypA and zraP are regulated by multiple bEBPs ( i . e . NtrC and at least one other ) , or that bEBPs other than NtrC are activated under the growth conditions tested .', 'The latter is more likely since no NtrC binding was detected upstream of hypA or zraP by ChIP-seq [20] .', 'We also observed three transcripts induced under low nitrogen conditions that initiate from intragenic σ54 binding sites .', 'These σ54 binding sites , within nagB , yqeC and rlmD , are located immediately upstream of the start sites for the transcripts induced by nitrogen limitation , and the predicted -24 and -12 motifs are oriented in the same direction as the associated RNAs ( Fig 6B ) .', 'In each case , the RNA that appears to be transcribed by RNAP:σ54 extends through the adjacent gene ( Fig 6B ) .', 'Thus , these RNAs appear to be mRNAs with unusually long 5ʹ UTRs .', 'A recent study identified an NtrC-activated σ54 promoter inside rlmD [20] .', 'This promoter is an exact match to the one we identified , and drives transcription of the entire relA gene ( adjacent to rlmD ) , consistent with our analysis .', 'To further investigate the intragenic σ54 binding sites within nagB and yqeC , we constructed strains with epitope tags fused to 3ʹ ends of nagE or yqeB .', 'We then constructed derivatives of these strains with mutations in the σ54 binding site inside nagB , or yqeC , respectively ( one silent change and one His → Lys codon change in nagB; two silent changes in yqeC; Fig 7A ) .', 'We measured association of σ54 with the wild type and mutated sites using ChIP-qPCR .', 'Our data indicate that mutating the putative binding sites greatly reduces binding of σ54 , confirming that these are genuine σ54 binding sites ( Fig 7B ) .', 'The microarray data described above strongly suggested that each of these intragenic σ54 binding sites is a promoter for an mRNA for the downstream gene .', 'To test this hypothesis , we used qRT-PCR to measure mRNA levels of the downstream gene for each putative promoter ( nagE and yqeB ) in wild type cells and cells in which the binding site is disrupted ( Fig 7C ) .', 'These data indicate that mutation of either σ54 binding site results in a large decrease in the mRNA level for the downstream gene .', 'Lastly , we used Western blotting with an antibody specific to the epitope tags to measure NagE and YqeB protein levels in cells with wild type and mutant promoters .', 'Consistent with the qRT-PCR data , mutation of either promoter resulted in a decrease in the protein level for the downstream gene ( Fig 7D ) .', 'In the case of NagE , the decrease in protein level was modest ( ~2-fold ) , whereas YqeB was undetectable in the promoter mutant .', 'We conclude that the σ54 binding sites within nagB and yqeC represent promoters for nagE and yqeB , respectively , with the mRNAs having unusually long 5ʹ UTRs .', 'We speculated that other intragenic σ54 binding sites represent genuine promoters .', 'We therefore compared each site across a range of bacterial species , mostly from the family Enterobacteriaceae .', 'σ54 is well conserved across these species ( e . g . 62% identical , 79% similar amino acid sequence between E . coli and V . cholerae ) , suggesting that it binds with similar DNA sequence specificity .', 'Furthermore , ChIP-seq of σ54 in V . cholerae identified a very similar motif to the E . coli σ54 motif identified here ( Fig 1B ) [19] , despite the two species having diverged >600 million years ago [40] .', 'Therefore , we used a position weight matrix derived from our MEME analysis ( Fig 1B ) to score sequences , from other species , that correspond to homologous regions to those surrounding the 135 high stringency σ54 binding sites in E . coli .', 'In some cases , no homologous region was identified .', 'A summary of the conservation analysis is shown in Fig 8 , and a complete list of conservation scores is shown in S5 Table .', 'Our analysis indicated that , as a group , intergenic binding sites ( Fig 8A ) are better conserved than intragenic sites ( Fig 8B ) .', 'However , many intragenic σ54 binding sites are conserved , suggesting that these sites are functional .', 'Importantly , the computationally determined motif score correlates well with in vivo binding of σ54 ( Fig 8C ) : the Spearman’s Rank Correlation Coefficient for the “OS” class of binding site ( intergenic , oriented towards a gene ) is 0 . 67 , and for the rest of the binding sites is 0 . 38 .', 'The sequence-based phylogenetic analysis described above predicted that many of the σ54 sites we detected in E . coli are functionally conserved in Salmonella enterica .', 'To test this prediction , we used ChIP-qPCR to measure association of σ54 with 14 sites in Salmonella enterica serovar Typhimurium that were predicted on the basis of sequence conservation .', 'Only five of these sites are in the classical “outside sense” orientation ( intergenic , oriented towards a gene ) .', 'As a control , we performed ChIP-qPCR without antibody .', 'In all cases , we detected robust association of σ54 , dependent upon the presence of antibody in the ChIP ( Fig 9 ) .', 'We conclude that many intragenic σ54 binding sites are functionally conserved in S . enterica .', 'Moreover , these data validate the sequence-based predictions of conservation ( Fig 8 ) .'], ['Unlike members of the σ70 family , σ54 proteins can bind promoters , in the context of RNAP holoenzyme , in a transcriptionally inactive state .', 'Therefore , while σ54-dependent transcription will only occur under conditions that activate the relevant bEBPs , the binding of σ54:RNAP holoenzyme to promoter DNA is not condition-dependent .', 'Our work takes full advantage of this characteristic to map the σ54 promoters of the E . coli genome using ChIP-seq .', 'In contrast , we reasoned that genome-wide expression levels of most σ54-transcribed genes are unlikely to differ in cells lacking σ54 , since most bEBPs are inactive in standard growth media .', 'This is supported by our RNA-seq analysis , which identified only 5 σ54-transcribed RNAs ( Fig 5 and S4 Table ) , and by a genome-scale analysis of σ54 in V . cholerae [19] .', 'Only 20 σ54 promoters have previously been experimentally validated in E . coli [26] .', 'Using ChIP-seq , we have confirmed 19 of these and have vastly expanded the list of known σ54 binding sites to 135 .', 'Moreover , our ChIP-seq data analysis with a relaxed cut-off is consistent with >250 σ54 binding sites , based on those for which we identified a motif .', 'Our data indicate that , for the conditions we used , σ54 binds most or all sites in the context of transcriptionally inactive RNAP holoenzyme ( Figs 4 and 5 ) .', 'As discussed below , many of the novel sites are likely to be non-functional , but there is strong evidence that a substantial subset has conserved function .', 'In addition to the 19 previously described σ54 promoters verified by our work , we confirmed 15 predicted σ54 promoters .', 'Our data are inconsistent with most of the previous predicted σ54 promoters [26]; false negatives due to the conditions we used are possible , but unlikely since σ54 is expected to bind all DNA sites under all growth conditions in which σ54 is expressed .', 'We have also identified 8 novel σ54 binding sites in intergenic regions , close to an appropriately oriented gene ( Table 1 ) .', 'This brings the total of canonical σ54 binding sites , i . e . those that are intergenic and likely to represent promoters for the downstream gene , to 42 .', 'The identities of these genes support a role for σ54 in transcribing genes involved in a wide range of cellular functions .', 'The majority of the 135 high stringency σ54 binding sites we identified are located inside genes .', 'Moreover , eight of the intergenic σ54 binding sites are located far ( >300 bp ) from the nearest appropriately oriented gene ( Fig 3A ) .', 'A recent ChIP-chip study of σ54 in Salmonella enterica also suggested the existence of intragenic binding sites , although the resolution of that study was at the level of whole genes due to the microarray design ( PCR products for entire genes ) [14] .', 'A ChIP-seq study of σ54 in V . cholerae identified only 7 intragenic σ54 binding sites [19] , suggesting that the function of σ54 in some species may be more restricted .', 'This is consistent with our analysis of σ54 binding site conservation , which showed conservation of very few binding sites between E . coli and V . cholerae ( Fig 8 ) .', 'Only a few other alternative σ factors have been mapped using ChIP-chip or ChIP-seq [10 , 11 , 13 , 15 , 16 , 17 , 41 , 42] , and in most cases the large majority of reported binding sites were intergenic .', 'However , E . coli σ32 , σ28 , and Mycobacterium tuberculosis σF were reported to bind large numbers of intragenic sites [15 , 16 , 17 , 18] .', 'In all three cases , a few intragenic binding sites were associated with transcription of RNAs that initiate inside a gene , sometimes in the antisense orientation [15 , 16 , 18] .', 'Intragenic σ54 binding sites as a group have significantly lower σ54 occupancy , as measured by ChIP-seq , than intergenic binding sites ( Mann Whitney U Test p < 1e-8 ) .', 'This suggests that many intragenic σ54 binding sites represent “biological noise” , binding sites created by chance due to sequence constraints imposed by other factors such as coding sequence .', 'However , our data indicate that σ54 binding site function and the level of σ54 binding do not correlate well .', 'For example , σ54 binding sites within nagB , rlmD and yqeC are associated with transcription of mRNAs ( Figs 6 and 7 ) but have relatively low binding scores ( Fold Above Threshold scores ( see Methods ) of 8 , 2 and 7 , respectively ) .', 'Our bioinformatic analysis indicates widespread sequence conservation of many intragenic σ54 binding sites ( Fig 8 ) , strongly suggesting important biological functions for these sites .', 'In some cases , conservation is restricted to a few species .', 'Nonetheless , even conservation solely in S . enterica , one of the closest relatives to E . coli that we analyzed , is a strong indication of function , since E . coli and S . enterica diverged from a common ancestor ~100 million years ago [43] .', 'We compared the σ54 binding sites identified in our study to those identified by ChIP-chip in S . enterica [14] .', 'Some predicted σ54 binding sites in S . enterica are shared with those in E . coli .', 'In particular , 20 intergenic σ54 binding sites are common to both species ( Table 1 ) .', 'Only six intragenic σ54 binding sites described in S . enterica correspond to genes for which we detected an intragenic site in E . coli ( Table 2 ) .', 'However , this low degree of overlap is likely due to technical limitations of the ChIP-chip method on sensitivity and resolution .', 'Indeed , many more of the σ54 binding sites that we identified in E . coli are conserved at the sequence level in S . enterica ( Fig 8 ) .', 'Consistent with this , we detected association of σ54 with five such sites in S . enterica as well as four of the sites identified in the S . enterica ChIP-chip study ( Fig 9 ) .', 'Thus , many σ54 binding sites are not only conserved between E . coli and S . enterica at the sequence level , but are also functionally conserved .', 'It is possible that conservation of some σ54 binding sites could be explained by sequence constraints imposed by the amino acid sequence of the overlapping protein , e . g . for the binding site within the highly conserved gene rpmJ .', 'However , in many cases , the predicted level of σ54 binding is similar between species even though the DNA sequence changes .', 'For example , changes can occur at positions that do not contribute to σ54 binding ( e . g . position 9 in Fig 1B ) .', 'To specifically address this question , we compared conservation of “important” bases within intragenic σ54 sites ( i . e . those that rarely deviate from the consensus; positions 3 , 4 , 14 , 15 and 16 in Fig 1B ) to that of ‘unimportant” bases ( i . e . those with low information content in the σ54 motif; positions 1 , 8 , 9 , 10 and 12 in Fig 1B ) .', 'If the σ54 binding site is the subject of positive selection , the unimportant bases should be less well conserved than the important bases , reflecting the sequence requirements for σ54 binding .', 'I
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription .', 'There are two families of σ factor: the σ70 family and the σ54 family .', 'Members of the σ54 family are distinct in their ability to bind promoter DNA sequences , in the context of RNA polymerase holoenzyme , in a transcriptionally inactive state .', 'Here , we map the genome-wide association of Escherichia coli σ54 , the archetypal member of the σ54 family .', 'Thus , we vastly expand the list of known σ54 binding sites to 135 .', 'Moreover , we estimate that there are more than 250 σ54 sites in total .', 'Strikingly , the majority of σ54 binding sites are located inside genes .', 'The location and orientation of intragenic σ54 binding sites is non-random , and many intragenic σ54 binding sites are conserved .', 'We conclude that many intragenic σ54 binding sites are likely to be functional .', 'Consistent with this assertion , we identify three conserved , intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5ʹ UTRs .']
['Bacterial RNA polymerases must associate with a σ factor to bind to promoter DNA sequences upstream of genes and initiate transcription .', 'There are two families of σ factor: σ70 and σ54 .', 'Members of the σ54 family are distinct from members of the σ70 family in their ability to bind promoter DNA sequences , in association with RNA polymerase , in a transcriptionally inactive state .', 'We have determined positions in the Escherichia coli genome that are bound by σ54 , the archetypal member of the σ54 family .', 'Surprisingly , we identified 135 binding sites for σ54 , a huge increase over the number of previously described sites .', 'Our data suggest that there are more than 250 σ54 sites in total .', 'Strikingly , most σ54 binding sites are located inside genes , whereas only one intragenic σ54 binding site has previously been described .', 'The location and orientation of intragenic σ54 binding sites is non-random , and many intragenic σ54 binding sites are conserved in other bacterial species .', 'We conclude that many intragenic σ54 binding sites are likely to be functional .', 'Consistent with this notion , we identify three σ54 promoters in E . coli that are located inside genes but drive transcription of unusual mRNAs for the neighboring genes .']
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