- Research article
- Open Access
Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 (RpoN) regulon of Salmonella Typhimurium LT2
BMC Genomicsvolume 14, Article number: 602 (2013)
Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ54 regulon in a bacterium is that the σ54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ54-dependent promoters for the characterization of the σ54 regulon of Salmonella Typhimurium LT2.
The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ54 binding sites suggest that many of these sites are capable of functioning as σ54-dependent promoters.
Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ54-dependent promoters of the S. Typhimurium LT2 σ54 regulon under a single growth condition, this approach is likely to be valuable for examining σ54 regulons in other bacterial species. The S. Typhimurium σ54 regulon included a high number of intragenic σ54 binding sites/promoters, suggesting that σ54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
Transcription in eubacteria is mediated by the RNA polymerase holoenzyme (Eσ), which has five constant subunits (α2ββ’ω) and a variable subunit (σ). The constant subunits constitute the RNA polymerase core (RNAP), which has the polymerization activity; the σ subunit determines promoter recognition and functions in the Eσ-promoter transition from closed complex to open complex (isomerization). The primary σ factor in a bacterium, such as σ70 in Escherichia coli, controls transcription of most housekeeping genes in the cell; alternative sigma factors have specialized regulons that function in the response to environmental stressors or morphological changes, or in developmental systems (for review see ). In many bacteria the alternative σ factor σ54 (also called RpoN or NtrA) has unusually diverse regulons, with genes that function in a variety of cellular processes, including flagellar biogenesis, response to nitrogen starvation, transport and metabolism of carbon substrates, and tolerance to heavy metals [2–6].
Multiple features, including protein structure, promoter consensus sequence, and mode of activation, distinguish σ54 from all other primary and secondary sigma factors, which constitute the σ70 family (reviewed in [1, 7]). Although both σ54- and σ70-type sigma factors associate with the β and β’ subunits of RNAP and mediate the binding of Eσ to specific promoter sequences, σ54 differs extensively from σ70-type sigma factors in primary amino acid sequence and domain organization (reviewed in ). The essential promoter features for Eσ54 recognition and binding center around conserved GG and TGC elements at -24 and -12, respectively, relative to the transcription start site (TSS) , while holoenzymes with the various σ70-type sigma factors generally recognize and bind promoter elements at -35 and -10 with the consensus sequences TTGACA and TATAAT, respectively (reviewed in ). Perhaps the most important feature of Eσ54 that differs from Eσ70 is the isomerization process (Figure 1A). For Eσ70 the transition from closed complex to open complex is usually spontaneous and rapid, so regulation of transcription initiation frequently occurs at the level of closed complex formation. Initiation of transcription by Eσ54 more closely resembles eukaryotic Pol II systems in that Eσ54 forms a stable closed complex that requires a bacterial enhancer-binding protein (bEBP) and ATP hydrolysis for isomerization to open complex (reviewed in ). The bEBPs add an additional level of complexity to the σ54 regulon.
bEBPs have a modular structure that is generally conserved: an N-terminal regulatory domain, a central AAA+ ATPase domain, and a C-terminal DNA binding domain (Figure 1B; reviewed in ). These proteins activate transcription from σ54-dependent promoters in three basic steps (Figure 1A). First, the bEBP receives an environmental stimulus through phosphorylation, ligand binding, or protein-protein interactions with the N-terminal regulatory domain that stimulates the bEBP to multimerize through the AAA+ ATPase domain and bind to an upstream activator sequence (UAS or enhancer) via the C-terminal DNA binding domain. The bEBP-UAS complex is then brought into contact with the Eσ54-promoter closed complex via a DNA looping event and interactions between highly conserved regions of the AAA+ ATPase domain of bEBP and σ54. Finally, ATP hydrolysis drives isomerization, allowing the initiation of transcription.
The requirement for bEBP-mediated activation of σ54-dependent transcription presents two problems for global analysis of a σ54 regulon. The first is the need for the proper environmental stimulus to activate bEBPs. Since the Eσ54 closed complex requires an activated bEBP, σ54-dependent promoters are usually transcriptionally silent in the absence of the specific stimulus for the bEBP . Analysis of transcription from σ54-dependent promoters under any single growth condition would miss operons whose bEBPs are not activated under the condition tested. Secondly, the requirement for the UAS or enhancer by most bEBPs presents a challenge for predicting whether a Eσ54 binding site is functioning as a promoter or not. There is no common consensus sequence for the enhancer and their position relative to the promoter can be quite variable. For many σ54-dependent promoters the UAS sequence lies ~70-150 bp upstream of the promoter, but other configurations have been characterized, such as enhancers located 1.5 kb downstream of the rocG promoter in Bacillus subtilis and up to 3 kb upstream of the promoter in artificial constructs of the glnA operon from E. coli. If a σ54 binding site is examined for promoter activity in isolation, such as in a promoter-reporter vector, it is difficult to discern whether a site is inactive because it is not a promoter or because the enhancer was not included in the cloned sequence.
Previous studies to define the σ54 regulons of Escherichia coli, Vibrio cholerae and Geobacter sulfurreducens have recognized the limitations presented by the requirement for activated bEBPs in the characterization of the full σ54 regulon, even when σ54 is overexpressed from a heterologous promoter. Our approach to overcoming these problems in the global characterization of σ54 regulons in bacteria is the utilization of a constitutively-active, promiscuous bEBP, the AAA+ ATPase domain of Sinorhizobium meliloti DctD [16, 17]. We chose to assess the efficacy of this approach in Salmonella enterica subsp. enterica serovar Typhimurium LT2 (hereafter referred to as S. Typhimurium LT2), a widely-used laboratory strain, because it has a moderately-sized σ54 regulon with 13 known or predicted bEBPs , providing sufficient diversity in bEBPs to test our hypothesis.
We report here that use of this constitutively-active, promiscuous bEBP in DNA microarrays and promoter function assays permitted detection of nearly all known and predicted σ54-dependent operons. These studies also revealed a new σ54-dependent promoter expressing a putative cas1 gene in S. Typhimurium LT2 (STM2938). In addition, chromatin immunoprecipitation-microarray (ChIP-chip) analysis combined with bioinformatics identified 70 Eσ54 or σ54 binding sites, of which 41 appear to be within open reading frames (ORFs). This surprising number of intragenic sites suggests regulatory roles for σ54 or Eσ54 that may involve repression, transcriptional interference, or expression of cis- or trans-acting small RNA (sRNA) [19, 20].
Results and discussion
Utility of a promiscuous, constitutive bEBP in characterizing the σ54 regulon
Since all known σ54-dependent promoters require an activated bEBP for transcription initiation, it is a challenge to find a condition under which all promoters can be detected within the σ54 regulon of a bacterium. In the recent mapping of the S. Typhimurium SL1344 transcriptome using early stationary phase cultures in rich media (Lennox broth), only one of the known or predicted σ54-dependent gene transcripts was detected, pspA. The currently favored approach is over-expression of σ54 to facilitate detection of σ54-dependent promoters, which assumes a reasonable basal level of activation of the bEBPs. Using relatively low cutoffs for the fold-change (1.5- to 2-fold) in transcript levels between the σ54-overexpression strain and wild type or ΔrpoN strains, a considerable portion of the σ54-dependent transcriptome was defined in Escherichia coli, Vibrio cholerae and Geobacter sulfurreducens. However, not all previously-identified σ54-dependent operons were detected for E. coli and G. sulfurreducens, and evidence from the V. cholera and G. sulfurreducens studies suggests that overexpression of σ54 may repress expression from some σ54-dependent promoters and alter expression of σ54-independent promoters [13–15]. We hypothesize that a promiscuous and constitutive variant of the bEBP DctD from S. meliloti can activate transcription from all σ54-dependent promoters in S. Typhimurium LT2 at wild-type levels of σ54 under a single growth condition, thereby facilitating global characterization of the σ54 regulon without overexpression of σ54. This promiscuous and constitutive DctD variant is missing the N-terminal response regulator and C-terminal DNA binding domains, leaving only the central AAA+ ATPase domain, residues 141 to 390 of DctD and referred to hereafter as DctD250 . Previous work showed that DctD250 was able to interact with Eσ54 in E. coli to drive transcription from the chromosomal glnA promoter and from the S. meliloti dctA promoter in the absence of native DctD and without an enhancer sequence [16, 17].
The σ54-dependent promoters of S. Typhimurium LT2 are normally responsive to one or more of thirteen known and predicted bEBPs under various growth conditions , so to initially assess DctD250 activation of transcription from σ54-dependent promoters that respond to different bEBPs in Salmonella, the σ54-dependent promoters for the glnKamtB (STM0462) and rtcBA (STM3521) operons were introduced upstream of a promoter-less lacZ gene and the reporter plasmids were transformed into a derivative of S. Typhimurium LT2 (wild-type; WT) and WT containing the DctD250 expression plasmid (WT + DctD250) to perform β-galactosidase assays. The glnKamtB and rtcBA promoters were chosen because neither has predicted σ70-dependent promoters within the cloned promoter region and each is responsive to a different bEBP: NtrC for glnKamtB and RtcR for rtcBA. In the WT strain, the glnKamtB and rtcBA operon promoters expressed lacZ at very low levels; but in the presence of DctD250, lacZ was expressed at 150- and 16-fold higher levels, respectively (Table 1). To compare the level of expression stimulated by DctD250 to the level that is seen under physiological conditions that activate the promoter-associated bEBP, lacZ expression from the glnKamtB promoter was assayed in the WT strain in nitrogen-limiting medium, which activates NtrC. Under nitrogen-starvation conditions NtrC multimerizes, binds the enhancer in the cloned promoter region, and hydrolyzes ATP to stimulate transcription by Eσ54 at the glnKamtB promoter (see Figure 1A). In the presence of activated NtrC, the glnKamtB promoter expresses lacZ at a nearly 10-fold higher level than in the presence of DctD250. This reduced level of activation by DctD250 relative to the cognate bEBP under activation conditions is consistent with previous studies comparing the activity of truncated versions of bEBPs, which must interact with Eσ54 from solution, to that of the wild type bEBPs, which are directed to the target σ54 promoter via binding to the enhancer sequence [17, 24]. The control reporter plasmids pDV6, which has the σ70-dependent, circle junction promoter from IS492, and the promoter-less pDS12 expressed lacZ at approximately the same level in WT as WT + DctD250 (Table 1). Based on these results, DctD250 activates transcription from σ54-dependent promoters that are normally responsive to different bEBPs under different growth conditions. Therefore, we performed DNA microarray and promoter-reporter analyses in the presence of the promiscuous, constitutive activator DctD250 to assess the efficacy of this approach in defining the σ54 regulon of S. Typhimurium LT2.
Microarray analysis of σ54-dependent transcripts in Salmonella expressing DctD250
To determine the genes whose transcription is controlled by σ54 in S. Typhimurium LT2 we performed a microarray analysis comparing WT+DctD250 to an isogenic strain with a deletion of rpoN (ΔrpoN+DctD250). RNA collected during mid-log phase growth in nutrient medium was reverse transcribed and cDNAs from each strain were differentially labeled and applied to a complete ORF array containing all annotated open reading frames for S. Typhimurium LT2 . Open reading frames that were transcribed in WT at a level > 3-fold higher than in the ΔrpoN strain, with a p-value <0.02, were considered up-regulated and, for the purpose of the initial categorization of these results, an operon was considered up-regulated if at least one gene met these criteria. In three biological replicates, the same 33 operons were up-regulated in the presence of σ54. The microarray results for S. Typhimurium LT2 genes within operons that meet the criteria for up-regulation, or that are known or predicted to be σ54-dependent, are shown in Table 2 and Additional file 1. Only 4 genes, STM2722, STM2724, STM2729, and STM2730, which are part of 2 operons in the Fels-2 prophage, were down-regulated >3-fold with a p-value <0.02 in the WT strain as compared to the ΔrpoN strain.
Known σ54-dependent operons and sRNA
If our hypothesis is correct, then in the presence of DctD250 we should observe up-regulation of operons (one or more structural genes) and sRNA genes that are known to have σ54-dependent promoters, even though they are normally activated by different bEBPs. Previously, four Salmonella operons have been experimentally shown to be regulated by σ54: prpBCDE, glnHPQ, argT, and glnALG. Additionally, two sRNA genes, glmY and glmZ, have also been shown to have σ54-dependent promoters . Table 2 summarizes the genes, functions, bEBPs, and microarray results for the known σ54-dependent operons and sRNA genes of Salmonella.
The DNA microarrays showed up-regulation of all four known σ54-dependent operons in Salmonella, prpBCDE, glnHPQ, argT, and glnALG (Table 2). The two sRNA genes with known σ54-dependent promoters did not appear up-regulated by σ54. This result was not surprising since in S. Typhimurium both glmY and glmZ possess σ70-dependent promoters that fully overlap the σ54-dependent promoters, such that the Eσ70 and Eσ54 compete for binding to their respective promoters . Gopel et al.  demonstrated that the level of glmY transcription was similar in wild type and ΔrpoN cells and that transcription of glmZ actually increased in the rpoN mutant, reflecting that the σ70-dependent promoter for glmZ is stronger than the σ70-dependent promoter for glmY. The presence of a σ70 promoter does not necessarily preclude detection of a σ54-dependent promoter controlling expression of a gene or operon in these microarray assays, though; the promoter region of glnA has non-overlapping σ70- and σ54-dependent promoters , yet was up-regulated 48-fold. Taken together, these results for the known σ54-dependent promoters are consistent with our hypothesis that DctD250 can promiscuously and constitutively activate σ54-holoenzyme at a variety of σ54-dependent promoters.
Confirmation of predicted σ54-dependent operons
There are 20 operons that we define as ‘predicted’ σ54-dependent operons in Salmonella. These predictions are based on in silico analyses indicating either homology to known σ54-dependent operons in E. coli and other enteric bacteria or promoter sequence homology along with genetic proximity to predicted bEBP genes [3, 5, 18, 22, 23, 30–39]. However, σ54-dependent transcription of these operons has not previously been experimentally demonstrated in Salmonella. In the DNA microarrays, 16 of the 20 operons that have been predicted to have σ54-dependent promoters in Salmonella were up-regulated in WT+DctD250 as compared to ΔrpoN+DctD250 (Table 2), providing experimental evidence that these genes are, in fact, regulated by σ54 in S. Typhimurium LT2.
For these 16 up-regulated σ54-dependent operons there are 11 different bEBPs that either are known or predicted to activate expression from their σ54-dependent promoters (Table 2). Five of the up-regulated operons, STM0577-0572, STM0649.s-0653, STM2360-2356, STM3772-3766, and STM4535-4540.s, were predicted to be σ54-dependent based on linkage to a predicted bEBP and an upstream sequence with the essential -12 and -24 elements of a σ54-dependent promoter . There are no orthologs in E. coli for the predicted bEBPs associated with these operons; three of these predicted bEBPs, STM0571, STM3773 and STM4534, are similar to the LevR-type EBPs found in Gram-positive bacteria . In addition to the microarray evidence presented here for σ54 regulation of these operons, we know that STM3773 is the bEBP controlling expression of STM3772-3776 and that this operon encodes the components of a phosphotransferase system permease for D-glucosaminic acid and enzymes required for catabolism of this acid sugar . These results show that DctD250 can activate expression at σ54-dependent promoters that are normally regulated by the LevR-type bEBPs.
Of the four predicted σ54-dependent operons that did not fulfill our criteria for upregulation in the microarray, at least two have additional σ54-independent promoters, which may have masked the effect of σ54 on transcription levels. The heat shock sigma factor gene rpoH has been shown to be under the control of additional promoters and other regulatory proteins in E. coli. The conservation of this promoter region for rpoH in S. Typhimurium LT2 suggests that a similar complex regulatory scheme may be involved , thereby reducing the effects of the ΔrpoN mutation. The yeaGH operon, which was just below the 3-fold cutoff for up-regulation in the microarray analysis, has previously been shown to be under control of σS in Salmonella; however, our assays utilized S. Typhimurium LT2, which has a defective rpoS gene due to a transversion mutation in the start codon . The promoter-reporter assay with the yeaGH promoter region, described below, suggests there is a σ54- and σS-independent promoter expressing the yeaGH operon in both the WT+DctD250 and ΔrpoN+DctD250 strains.
The frequency of alternate promoters seen for the σ54-dependent operons in Salmonella (at least 15% for the known and predicted promoters in our analyses) is not unique. Zhao et al.  estimate that 14% of σ54-dependent genes in E. coli are transcribed by σ70-associated RNA polymerase and suggest that expression of σ54–dependent genes from alternate promoters allows for differential expression under various environmental conditions.
New potential σ54-dependent genes
In addition to the σ54-dependent expression of known or predicted genes and operons, the DNA microarray analysis revealed up-regulation of a gene, STM2938, which has not previously been reported or predicted to be σ54-dependent. STM2938 is the penultimate gene in a nine-gene operon that is annotated as a group of CRISPR-associated (cas) genes. Although none of the other genes in this operon seem to be controlled by σ54, further evidence is presented below that supports the presence of a σ54-dependent promoter within the gene upstream of STM2938. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and cas genes constitute an adaptive immune system in bacteria and archaea that protects against invading mobile DNA, such as phage and plasmids . The response to phage infection, which is referred to as phage shock, is regulated by σ54 and the bEBP PspF in E. coli; thus, it would not be surprising for essential components of the bacterial immune response in phage infection to be regulated similarly. The potential σ54-dependent gene STM2938 is a homologue of the cas1 gene, which is an endonuclease that is associated with all CRISPR loci and is most likely involved in the adaptation phase of the CRISPR-cas immune system . The regulation of this cas1-like gene by PspF in Salmonella is currently under investigation.
There were 12 additional ORFs that met the 3-fold cutoff for up-regulation by σ54 in the microarray assay, including genes for pilin biosynthesis (hofB), histidine ammonia lyase (hutH), bEBPs (ygaA, fhlA), propanediol utilization (pduG), siderophore production (iroD), and cell invasion (invG). The whole genome chromatin immunoprecipitation assays described below did not reveal σ54 binding sites associated with these ORFs; thus the expression of these genes may be indirectly affected by the absence of σ54 in the ΔrpoN mutant, or constitute false positives (Additional file 1).
ChIP-chip analysis of genome-wide σ54 binding sites in Salmonella
In the characterization of the σ54 regulon of Salmonella, determination of the genomic binding sites for the Eσ54 allows confirmation of primary transcripts indicated by microarray analysis and recognition of potential σ54-regulated genes that might not have been detected due to instability of the transcripts. To assess the binding of Eσ54 in the S. Typhimurium LT2 genome, we isolated σ54-DNA complexes from WT and ΔrpoN strains that did not contain the DctD250 expression plasmid. Since bEBPs do not activate transcription by recruiting Eσ54 to promoter sequences , inclusion of DctD250 should not be necessary to detect binding of holoenzyme to promoter sequences in the ChIP-chip assay. Protein-DNA complexes containing either Eσ54 or σ54 are pulled down in the ChIP with α-σ54. The σ54 subunit is most likely to interact with the genome in the context of the RNA polymerase holoenzyme; however, σ54 has been shown to specifically bind in the absence of the core RNA polymerase at σ54-dependent promoters that have a T-tract upstream of the GC in the -12 promoter element . DNA fragments from the α-σ54 ChIP were labeled and applied to the same complete open reading frame arrays as used in the microarray analysis.
Since the use of the ORF arrays did not allow direct mapping of the binding sites, we combined the ChIP-chip data with in silico analysis to determine the potential σ54 DNA binding sites. A Position-Specific Score Matrix (PSSM) was created using 27 known or previously predicted σ54-dependent promoters from S. Typhimurium LT2 (Additional file 2); the extent of each promoter sequence used for the PSSM (18 bp) was based on the consensus sequence for σ54-dependent promoters defined by Barrios et al.  and comparison analysis of the known Salmonella σ54-dependent promoters. This PSSM was applied with the Motif Locator program  to the enriched ORF sequence and 1000 bp of flanking sequence on both sides of the ORF to identify potential σ54 DNA binding sites. The size range of DNA fragments that were pulled down via ChIP and amplified by ligation-mediated PCR was 200–1000 bp long, as determined by agarose gel electrophoresis, suggesting that intergenic binding sites up to 1000 bp from the enriched ORF might be detected in the ChIP-chip assays.
σ54 binding to promoters for known, predicted, and novel σ54-dependent operons
In the ChIP-chip assays with the WT and ΔrpoN strains, the promoter-proximal gene for all the 24 known and predicted σ54-dependent operons and the 2 sRNA genes (Table 2) were enriched, as defined by a stringent cut-off, i.e. signal ratio ≥3 and p-value <0.02 (Table 3). The associated promoter sequences, as determined by the in silico analysis, had PSSM scores ranging from 10.9 to 23.6 and were within 27 to 154 bp of the enriched ORF. In the DNA microarrays, six of the known or predicted σ54-dependent operons did not appear up-regulated; but in the ChIP-chip assays the promoter regions for all six operons gave signal ratios ranging from 3.2- to 39-fold greater in WT than in ΔrpoN cells. The detection of the σ54-dependent promoters for all the other known and predicted σ54-dependent operons supports the efficacy of our approach to mapping potential σ54-binding sites.
The ChIP-chip analyses also showed that only one (STM2938) of the 13 newly-identified, potential σ54-dependent operons from the DNA microarray assays has a σ54 DNA binding site associated with it (Table 3), suggesting that the other 12 operons may be indirectly regulated by σ54. The σ54 DNA binding site associated with STM2938, the cas1-like gene, is within the upstream gene, STM2939 (539 bp from the start of STM2938). Further characterization of this potential σ54 promoter is described below in the promoter-reporter analysis.
σ54 binding to newly identified potential promoter and regulatory sites
In total, 70 ORFs were each found to be enriched in 3 replicate samples for the WT cells as compared to the ΔrpoN cells in the ChIP-chip assays (Table 3). The potential σ54 binding site with the highest PSSM score within each enriched ORF or up to 1000 bp of flanking intergenic sequence was identified by Motif Locator and is reported in Table 3. For the 70 enriched ORFs, 29 of the associated binding sites mapped to intergenic regions and 41 of the potential σ54 binding sites were located within the enriched ORF (Figure 2). In determining the most likely binding site for an enriched ORF, sequence within an adjacent non-enriched ORF was not considered for potential σ54 binding sites, since the ORF containing the binding site should be enriched; therefore, even if a site with a higher PSSM score was located in an immediately adjacent non-enriched ORF, the next highest scoring site found in either the enriched ORF or adjacent intergenic sequence was reported as the potential binding site in Table 3. This reflects a limitation of the in silico prediction of σ54 binding sites based on a PSSM that was created with known and predicted intergenic promoter sequences; the sequences for intragenic promoters or for σ54 binding sites that are regulatory sites, but not promoters, may differ enough to appreciably affect PSSM scores.
Consensus sequences were generated using WebLogo  for the intergenic and intragenic potential σ54 binding sequences and for the promoter sequences used to generate the PSSM (Figure 3). Noteworthy differences in the consensus sequence for the intragenic σ54 binding sites, as compared to the consensus sequences for the intergenic σ54 binding sites and PSSM promoters, are at the −23 and −11 positions, which each contribute in different ways to σ54-promoter DNA interactions. The −23 A-T base pair is important in promoter recognition by σ54; the winged helix-turn-helix DNA binding motif of σ54 makes base-specific contacts with the top strand GG at positions −26 and −25 and with the bottom strand T at position −23 . The base pairs immediately adjacent to the conserved GC element in the -12 region of the promoter are involved in Eσ54 binding to form the stable closed complex; the bases on the bottom strand of the promoter at the -12 and -11 positions interact with σ54 in a short region of ‘early melting’ that stabilizes closed complex until the bEBP binds σ54 and activates the holoenzyme to transition to open complex . The reduced conservation of nucleotide sequence at the −23 and −11 positions for the potential intragenic σ54 binding sites may reflect varied functionality of these intragenic sites, or a level of inaccuracy inherent to in silico prediction of the binding sites associated with enriched ORFs in the ChIP-chip assays.
The position and orientation for each potential σ54 binding site are indicated in Table 3 and summarized for all the binding sites in Figure 2. This information is useful in considering possible functions for the binding sites. For example, the 16 intragenic σ54 binding sites oriented in the opposing direction of the gene might regulate by transcription interference and/or anti-sense RNA . Four intragenic σ54 binding sites are within 250 bp of the 5’ end of a downstream gene, or a large intergenic region (>100 bp), and oriented in the direction such that they might act as promoters for the downstream gene or a sRNA . Binding sites located near a functional σ54 promoter may serve to accelerate the search for the promoter by Eσ54 sliding from the secondary sites ; while binding sites adjacent to, or overlapping, a σ70- or σ54-promoter may bind Eσ54 or σ54 and repress or activate transcription from the other promoter . The possible functions of the σ54 binding sites are quite varied and many are dependent on whether the binding site can function as a promoter.
It is likely that our initial approach to defining the global binding sites of σ54 in S. Typhimurium LT2 resulted in an underestimation of the number of binding sites. Multiple sites within ~2,000 bp of an ORF would enrich one or two adjacent ORFs and, in our analysis, would have been counted as one site. In addition, since Eσ54-promoter closed complexes are reversible , some complexes might have not been detected due to high disassociation rates; detection of these sites may be improved in the presence of DctD250, which stimulates conversion of closed complex to the more stable open complex, and rifampicin, which prevents extension of RNA past the second or third nucleotide , thus improving the chances of cross-linking Eσ54 at the promoter sequence .
Promoter-reporter analysis to determine activity for predicted promoter sequences
To assess the functionality of σ54 binding sites defined by the ChIP-chip and PSSM analyses, promoter-lacZ fusion assays were performed using several of these sequences. We had two goals in performing these assays. First, we wanted to further confirm the σ54-dependent promoter activity for some of the predicted σ54-dependent promoters that were up-regulated in the DNA microarrays and enriched in the ChIP-chip assays. Secondly, we wanted to test the σ54 binding site predictions from the ChIP-chip combined with PSSM analyses; i.e. does a potential σ54 binding site equate to a σ54-dependent promoter? This promoter function assay is the initial exploration of the roles for σ54 binding sites located within intragenic regions.
Potential promoters were introduced upstream of a promoter-less lacZ gene in a reporter vector, either pDS11 or pDS12 (which differ only in their MCS sequence). These promoter-reporter plasmids were co-transformed along with the DctD250 expression plasmid into either WT or ΔrpoN cells. After induction of DctD250 expression, standard β-galactosidase assays were performed. The results from WT were compared to those from the ΔrpoN mutant to determine whether activity seen was σ54-dependent (Figure 4). For intergenic sequences that were known or predicted to be σ54-dependent promoters, the results matched those observed in the DNA microarray assays (Figure 4A, Table 2). The glnA, glnK, and STM3521 (rtcBA operon) promoters showed strong σ54-dependent activity. For the glmY, glmZ, rpoH, and yeaG promoters, transcription in the ΔrpoN mutant was either as high as or higher than in wild type cells. This is likely due to the presence of σ70-type promoters in the cloned sequence. In addition to the σ54 dependent promoters, other promoters have been reported upstream of glmY, glmZ, rpoH, and yeaG[28, 36, 41].
A total of eight intragenic sites identified in the ChIP-chip assay were selected for functional analysis (Figure 4B). All of these predicted sites had PSSM scores >10. As shown in Figure 4C, the sites chosen represent a variety of configurations with regard to their position and orientation within the ORF as well as the position and orientation of downstream ORFs. Given the possible functions for an intragenic promoter sequence (e.g. promoter for a downstream gene or sRNA, generation of antisense RNA, etc.), results of our analysis allow us to determine which, if any, of these roles may be attributable to any of these promoters.
Comparing the levels of lacZ expression in wild type cells to those in ΔrpoN mutants, we found that four of the eight intragenic sites were able to function as σ54-dependent promoters. For these sites, the difference observed between WT and ΔrpoN cells varied from 4.6-fold for the sequence located in STM2957 to 8.9-fold for the sequence within STM2939. Overall, the level of transcription from these promoters was relatively low, with Miller units ranging from ~30-100. The low activity levels may indicate that Eσ54 has a low affinity for these sequences or that the DctD250 is inefficient in productively engaging closed complexes formed at these sites. A subset of the promoter-reporter plasmids with intragenic sites that exhibited σ54-dependent transcription were also assayed in WT cells versus WT-DctD250 to determine the dependence of transcription on the promiscuous, constitutive bEBP (Table 1). All three intragenic promoters assayed, STM0699, STM2430, and STM2939, gave low levels of β-galactosidase activity in the absence of DctD250 and from 4.7- to 34-fold higher levels of β-galactosidase activity in the presence of DctD250. The σ54-dependent transcription from intragenic binding sites suggests previously unrecognized regulatory functions for σ54 in Salmonella; however, it will be critical to characterize transcription from their chromosomal loci before biological functions can be ascribed.
Some of the potential promoter sequences that were assayed failed to show any transcriptional activity. There are a number of possible reasons for the lack of promoter activity for these sites. Two likely explanations are: 1) the wrong sequence was chosen as the binding site based on the PSSM score and proximity to the enriched ORF in the ChIP-chip assays, i.e. a lower scoring sequence near the enriched ORF was the actual σ54 binding site; or 2) the σ54 binding site does not function as a promoter but serves another regulatory role, such as an operator site for regulating promoter activity, a site for transient binding in facilitated diffusion, or a site for sequestering Eσ54 in order to increase local concentration (since σ70 has a higher affinity for RNAP ).
Summary of S. Typhimurium LT2 σ54 regulon and comparison to σ54 regulons of other bacteria
Figure 5 summarizes the results from the DNA microarray and promoter-fusion assays performed in the presence of DctD250 and ChIP-chip in the absence of DctD250 to characterize the σ54 regulon of S. Typhimurium LT2. Based on DNA microarray, there are 33 up-regulated operons (76 genes; Additional file 1); global ChIP-chip combined with in silico analysis revealed at least 70 σ54 binding sites (Table 3), of which 21 were associated with up-regulated operons from the DNA microarrays. The promoter-lacZ fusions with seven of the 29 intergenic σ54 binding sites and eight of the 41 intragenic σ54 binding sites showed DctD250- and σ54-dependent expression for three intergenic sites (associated with up-regulated operons) and four intragenic σ54 binding sites (Table 1, Figure 4). The cellular functions impacted by genes in the σ54 regulon of S. Typhimurium LT2 are quite diverse, ranging from carbon-source and amino acid metabolism to response to stressors, such as nitric oxide and toxic levels of zinc (Table 2). Our results suggest that a new cellular process may be added to this extensive list—cell immunity through the CRISPR system; the role of σ54 in regulating a cas1-related gene within an operon of CRISPR-associated genes is presently being investigated.
The σ54 global regulon of S. Typhimurium LT2 may differ from that of virulent S. Typhimurium isolates due to accumulated mutations in this extensively-used, laboratory strain, particularly the rpoS mutation that contributes to attenuation of the LT2 strain . Changes in the level of expression of one sigma factor can alter the expression of genes that are expressed by different sigma factors ; for example, it has been shown that deletion of rpoN alters expression of σS-dependent promoters in E. coli. We are currently characterizing the σ54 global regulon of the virulent strain S. Typhimurium 14028s.
The σ54 regulons in other δ/γ-proteobacteria have been characterized experimentally to varying extents [13–15, 58–60]. Only in Vibrio cholera 037 strain V52 have both global transcripts and binding sites been characterized experimentally . In E. coli MG1655 and Geobacter sulfurreducens, the global σ54 transcriptomes were determined and local σ54 binding sites associated with up-regulated genes were assessed by computational analysis and selected promoters were assessed experimentally [13, 15]. The number and diversity of the operons that are directly controlled by σ54-promoters in these δ/γ-proteobacteria are comparable to that of S. Typhimurim LT2. The greatest variability in the σ54 regulons of the γ-proteobacteria appears to be the location of σ54 binding sites. Zhao et al.  estimated 70 σ54 promoters in E. coli MG1655, of which 13 (18%) were intragenic or located between convergently transcribed genes. In V. cholera, Dong and Mekalanos  identified a total 68 σ54 binding sites, of which 35 (51%) were intragenic and, similarly, we found 70 potential σ54 binding sites of which 41 (58%) appear to be located in intragenic regions.
Does the success with DctD250 in characterizing the S. Typhimurium σ54 regulon predict utility of this constitutive, promiscuous activator in defining σ54 global regulons in bacteria from other classes in the Proteobacteria phylum, or from other phyla? The key to activation of Eσ54 by DctD250 in diverse bacteria is the ability of the activator to make the appropriate interactions with σ54 in the context of the Eσ54-promoter closed complex; thus, comparison of interacting regions of σ54 and bEBPs between S. Typhimurium and phylogenetically diverse bacteria is a good predictor of success. Extensive characterization of bEBP activation of Eσ54 in closed complex has shown that the GAFTGA motif of the AAA+ ATPase domain plays a primary and essential role for productive interactions with Eσ54, which lead to transcriptional activation (reviewed in ); the GAFTGA motif is very highly conserved among bEBPs in all bacteria that encode σ54, which includes bacteria from a majority of the eubacterial phyla . It has not yet been determined which specific residues of σ54 are contacted by Loop 1 of the bEBP AAA+ ATPase domain, but it has been clearly demonstrated that multiple residues within the amino-terminal 50 amino acids of σ54 (Region I) are key determinants for activator interaction  and there is extensive conservation of amino acid sequence in Region I for σ54 from phylogenetically diverse bacteria . Thus, the comparison of interacting regions of σ54 and the AAA+ ATPase domain among diverse bacteria predicts that DctD250 will be a valuable tool in characterizing the σ54 regulons in many bacteria.
The results of DNA microarray and promoter-lacZ fusion analyses of the σ54 regulon of S. Typhimurium LT2 in the presence of DctD250 support our initial hypothesis: the AAA+ ATPase activation domain of DctD can stimulate transcription from σ54-dependent promoters in a constitutive and promiscuous manner, thereby facilitating the global characterization of σ54 regulons. Sixteen previously predicted σ54-dependent operons were confirmed, and a new σ54-dependent gene, cas1, was identified by the DNA microarray and ChIP-chip analyses. In addition, the ChIP-chip analyses indicate an excess of σ54 binding sites compared to the number of σ54-dependent transcripts and a high percentage of intragenic binding sites, suggesting that Eσ54 and σ54 may have more regulatory functions than transcription initiation at the start of an operon or sRNA. The number of functional promoters located inside genes suggests a need to consider such promoters in bioinformatic analyses of transcription factor binding sites.
Bacterial strains, media, and enzymes
The parental strain, designated wild-type, in these experiments was Salmonella enterica subspecies enterica serovar Typhimurium LT2 derivative MS1868 [leuA414(Am) hsdSB(r-m+)Fels-] . An isogenic derivative, TRH134, has a deletion in rpoN (ntrA) from codons 8 through 455, rendering it auxotrophic for glutamine . S. Typhimurium strains were cultured in either nutrient broth (NB; Difco Laboratories), MOPS minimal media , or nitrogen-limiting MOPS . Media supplement concentrations were 5 mM L-glutamine (Gln), 40 μg/ml L-Leucine (Leu), and 10 mM L-glutamate (Glu). Cloning procedures were performed in E. coli DH5α cultured in Luria-Bertani medium (LB; Fisher Scientific). All strains were grown at 37°C. Antibiotics (Sigma-Aldrich) were used at the following concentrations (μg/ml) for E. coli/S. Typhimurium (NB)/ S. Typhimurium (MOPS), respectively: ampicillin (Amp) 80/120/50; spectinomycin (Spc) 50/125/50; streptomycin (Str) 25/75/0. All enzymes were purchased from New England Biolabs, unless otherwise indicated, and were used according to manufacturer’s recommendations.
Plasmid pPBHP92 is a derivative of the expression vector pTrcHisC (Invitrogen) that expresses the Sinorhizbium meliloti DctD AAA+ ATPase domain (E141-S390, designated DctD250) with an N-terminal 6x-His tag. This plasmid was constructed by digestion of pHX182  with NdeI, filling in the 5’-overhang with T4 DNA polymerase and subsequent digestion with XhoI. The blunt-XhoI fragment containing the truncated dctD was cloned into pTrcHisC, which had been cut with NheI, blunt-ended, and cut with XhoI. The truncated dctD is under control of Ptrc and subject to repression by the vector-encoded LacI. The reporter plasmids used in these studies, pDS11 and pDS12, are both derivatives of pDV6  that contain a promoter-less copy of lacZ downstream of a MCS region. The MCS region was generated by annealing two oligonucleotide primers (Additional file 3) which were then ligated into a pDV6 backbone that had been digested with BamHI and HindIII. pDS11 and pDS12 differ only in MCS sequence. Potential promoter sequences were amplified from S. Typhimurium LT2 genomic DNA using Taq polymerase and the primers in (Additional file 3) and cloned into pCR2.1 (Invitrogen). Sequencing analysis to determine accuracy and orientation was performed for all plasmids by Genewiz, Inc. (South Plainfield, NJ). Depending on their orientation in pCR2.1 potential promoter sequences were sub-cloned into pDS11/12 using XbaI and either KpnI or HindIII. Plasmid pTG4, which encodes the DctD AAA+ ATPase domain under control of Ptac/lacIq, was created by amplifying the corresponding region of pPBHP92 using primers DctD-F/R (Additional file 3), digesting the product with BamHI and HindIII, followed by ligation into the similarly digested pKH66 .
Transcriptional profiling by microarrays
S. Typhimurium strains MS1868 and TRH134, each bearing plasmid pPBHP92 (WT+DctD250 and ΔrpoN+DctD250, respectively), were grown overnight at 37°C in NB-Amp. Cultures were sub-cultured in fresh medium and grown to mid-log phase (OD600 ≈ 0.8). Since the basal level of DctD250 expression from pPBHP92 was shown to optimally activate transcription from a σ54-dependent dctA’-‘lacZ reporter , IPTG induction was not used for these cultures. RNA isolated using the RNAeasy kit (Qiagen) was used to generate differentially labeled cDNA using reverse transcriptase as previously described . Labeled cDNA was hybridized to DNA microarrays containing complete open reading frames (ORFs) from S. Typhimurium LT2 printed in triplicate . Microarrays were scanned with a ScanArray Lite laser scanner (Packard BioChip Technologies, Billerica, MA) using ScanArray Express 1.1 software. Signal intensities were quantified using QuantArray 3.0 (Packard). The ratio of WT+DctD250 signal to ΔrpoN+DctD250 signal was determined for each of the triplicate spots and the median value for each ORF was used in the statistical analysis . Data shown is the result of three biological replicates with statistical analysis performed using the WebArrayDB program [71, 72]. The intensity values for the three biological replicates of WT+DctD250 and of ΔrpoN+DctD250 were compared for the calculation of the p-values, where the null hypothesis was that the intensities for WT+DctD250 and ΔrpoN+DctD250 would be equivalent. Genes that displayed a WT+DctD250/ΔrpoN+DctD250 signal ratio of >3-fold with a p-value of <0.02 were considered to be up-regulated.
Chromatin immunoprecipitation (ChIP)
ChIP was carried out using the ChIP Assay kit (USB Corporation) essentially as described by the manufacturer’s instructions. Briefly, 100 ml cultures of S. Typhimurium strains MS1868 (WT) and TRH134 (ΔrpoN) were grown overnight in NB at 37°C and sub-cultured in fresh medium the next day. Once cultures reached mid-log phase (OD600 ≈ 0.7), cells were treated with formaldehyde (3 ml of a 37% solution per 100 ml of culture) for 10 min. at room temperature to cross-link proteins to DNA. Cross-linking was quenched by the addition of glycine (10 ml of 1.33 M solution per 100 ml of culture) and incubation at 4°C for 30 min. Cells were harvested, washed and lysed in accordance with kit instructions. Cells were lysed in two passages through a French pressure cell at 10,000 psi. Cell extracts were clarified and pre-cleared with the provided protein A-Sepharose bead slurry per the kit instructions. 0.6 ml of the resulting extracts were mixed with 2 μl of rabbit anti-serum against S. Typhimurium σ54 and incubated with gentle shaking overnight at 4°C. The next day, 50 μl of protein A-Sepharose bead slurry was added to each sample, incubated 1 hr at room temperature and collected by centrifugation. The beads were washed, and protein-DNA complexes were eluted from the beads and disrupted per the supplier’s instructions. DNA was purified from each sample using the Qiagen PCR purification kit.
Purified ChIP DNA was amplified by ligation-mediated PCR, adapting the procedure found at [http://www.flychip.org.uk/protocols/archive_protocols/lm_pcr.php]. Linkers consisting of complementary oligonucleotides (LM-PCR; Additional file 3) were ligated to the ends of purified DNA repaired with T4 DNA polymerase. Ligated was purified using the Qiagen PCR purification kit and the DNA was amplified with Taq polymerase (Fermentas; Burlington, ON) using LM-PCR-R as the PCR primer and the following cycling conditions: 55°C—2 min (1×); 72°C—5 min (1×); 94°C—5 min (1×); 94°C—1 min, 55°C—1 min, 72°C—1 min (24×); 72°C—5 min (1×); 4°C—hold. The resulting amplicons, most of which were 300–800 bp, were purified using the Qiagen PCR purification kit and to prepare dye-labeled DNA (Cy3 or Cy5) for hybridization to the S. Typhimurium complete ORF microarray. Microarrays were scanned and analyzed as above. ChIP-chip was performed on three biological replicates for the WT and ΔrpoN strains; the statistical analysis of the data was performed as described for the microarray data.
Identifying candidate σ54 binding sites in the S. Typhimurium genome
The Motif Locator program [http://www.cmbl.uga.edu/software.html] was used to identify candidate σ54 binding sites. The program applies the standard position-specific score matrix (PSSM) described in . We used a PSSM derived from the alignment of 27 high-confidence sites supported by experimental evidence in either Salmonella or E. coli (Additional file 2). Background nucleotide frequencies were assigned in accordance with the genomic G+C content. Pseudo-counts equal to the background frequencies were used in PSSM construction. For ORFs discovered in the ChIP-chip assay, this matrix was used to determine the most likely binding site either within the ORF itself or in the region ±200, 500, or 1000 bp surrounding the gene.
The DctD250 expression plasmid pTG4 was introduced into S. Typhimurium MS1868 and TRH134 by electroporation using a GenePulser 2 system (BioRad; Hercules, CA) and the resulting transformants were electroporated with pDS11 or pDS12 reporter constructs containing potential σ54-dependent promoter sequences. Overnight cultures grown in MOPS-LeuGln, or nitrogen-limiting MOPS-Glu, with the appropriate antibiotics were sub-cultured into fresh medium, grown to OD600 ≈ 0.2, and induced with 50 μM IPTG (empirically determined IPTG concentration for optimal expression of DctD250 from pTG4 to activate known σ54-dependent promoters on the reporter plasmids). Cultures were induced for 6 hours and β-galactosidase activity was measured as described previously  with the following changes: 1) assays were performed at 37°C, and 2) after stopping reaction, samples were centrifuged and OD420 of the supernatant was measured, eliminating the OD550 correction for cell debris. Activity was calculated as Miller units: [(OD420 × 1000)/(OD600 × Time(min) × volume (ml))] . Ratios of activity in wild type/ΔrpoN cells were compared and analyzed using a 2-tailed Student’s T-test. Data shown for each promoter construct represents ≥3 biological replicates.
Accession number for microarray and ChIP-chip data
The DNA microarray and ChIP-chip data were deposited in NCBI GEO under accession number GSE25849.
Bacterial enhancer-binding protein
RNA polymerase holoenzyme
Upstream activation sequence.
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We would like to thank the following people for their assistance on this project: Ashley Bono, Uchenna Ewulonu, and Trevor Wright generated reporter constructs. Tanya Grancharova sub-cloned DctD250. Sonya Chelliah performed ChIP pull-downs. Jennifer Turpin prepared and applied cDNA to the microarrays. This work was supported by National Science Foundation Grant MCB-1051175 (to T.R.H. and A.C.K.). MM and SP were supported in part by NIH Contract No. HHSN272200900040C from PATRIC and grants AI039557 AI052237, AI073971, AI075093, AI077645 AI083646, USDA grants 2009-03579-30127 and 2011-67017-30127, the Binational Agricultural Research and Development Fund. JM was supported in part by the National Science Foundation grant DBI-0950266.
The authors declare that they have no competing interests.
DS harvested RNA, carried out promoter-reporter assays, and drafted and revised and prepared the manuscript. JF prepared cDNA and preformed DNA microarrays assays and applied DNA from the ChIP to the microarrays. SP and MM produced the microarrays and protocols and performed the statistical analysis of the microarray therein. JM performed the bioinformatic analyses. TH conceived, designed, and coordinated the study, harvested RNA, and performed ChIP pulldowns. AK conceived, designed, and coordinated the study, harvested RNA, and drafted and revised the manuscript. All authors read and approved the final manuscript.