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BMC Genomics

Open Access

Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 (RpoN) regulon of Salmonella Typhimurium LT2

  • David J Samuels1,
  • Jonathan G Frye2,
  • Steffen Porwollik3,
  • Michael McClelland3,
  • Jan Mrázek1, 4,
  • Timothy R Hoover1 and
  • Anna C Karls1Email author
BMC Genomics201314:602

https://doi.org/10.1186/1471-2164-14-602

Received: 19 April 2013

Accepted: 28 August 2013

Published: 5 September 2013

Abstract

Background

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.

Results

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.

Conclusion

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.

Keywords

Sigma54RpoNBacterial enhancer-binding proteinRegulonSigma factor Salmonella

Background

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 [1]). 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 [26].

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 [8]). 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) [9], 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 [1]). 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 [10]). The bEBPs add an additional level of complexity to the σ54 regulon.
Figure 1

Activation of σ 54 -dependent transcription and activator structure. A) σ54 (red subunit) directs binding of the RNA polymerase (dark blue subunit) holoenzyme (Eσ54) to the -12, -24 promoter elements (light blue box). This closed complex is stable and cannot transition to open complex. In response to an environmental or cellular signal, the activator (bEBP; yellow dimers) oligomerizes. For most bEBPs, the oligomer binds to an enhancer (green box) 80 to 150 bp upstream of the promoter and DNA looping brings the activator in contact with σ54 in the Eσ54 closed complex. Hydrolysis of ATP by bEBP causes remodeling of Eσ54, which leads to open complex formation and transcription. There are a few bacteria with bEBPs that are missing the DNA binding domain; after oligomerization, these activators can bind to Eσ54 in closed complex with any promoter to stimulate open complex formation (promiscuous activation). B) The domain structure for the Sinorhizobium meliloti bEBP, DctD, is typical of most bEBPs. The amino-terminal regulatory domain (dark blue box) inhibits assembly of the bEBP oligomer until it interacts with an activation signal; the AAA+ ATPase domain (red box) mediates ATP binding and hydrolysis, as well as the protein-protein interactions between bEBPs (oligomerization) and between bEBP and σ54; the carboxyl-terminal DNA binding domain (aqua box) contains a helix-turn-helix motif for binding the enhancer. The truncated DctD variant, DctD250, is missing the regulatory and DNA binding domains, so that it is constitutively active and promiscuous in stimulating transcription from σ54-dependent promoters.

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 [8]). 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 [8]. 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[11] and up to 3 kb upstream of the promoter in artificial constructs of the glnA operon from E. coli[12]. 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[13], Vibrio cholerae[14] and Geobacter sulfurreducens[15] 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 [18], 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[21]. 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[13], Vibrio cholerae[14] and Geobacter sulfurreducens[15]. 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 [1315]. 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 [17]. 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 [18], 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[22] and RtcR for rtcBA[23]. 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[25], 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.
Table 1

DctD250-dependent activity of predicted and potential σ 54 -dependent promoters

  

Miller unitsb

 

Promotera

WT

WT+DctD250

WT-N

Intergenic:

   

STM0462 (glnKamtB)

1.1 ±0.1

180 ±22

1778 ±28

STM3521 (rtcBA)

14 ±6.8

220 ±55

N.D.

Intragenic:

   

STM0699

5.3 ±1.6

56 ±6.2

N.D.

STM2430

19 ±3.8

90 ±5.5

N.D.

STM2939

2.2 ±1.4

76 ±17

N.D.

Controls:

   

IS492-CJ (pDV6)

6400 ±2200

9000 ±4100

N.D.

Empty vector (pDS12)

2.0 ±0.5

8.4 ±3.5

4 ±0

a Promoters assayed in lacZ-reporter plasmids pDS11 and pDS12: intergenic promoters are predicted promoters for the glnALG, glnKamtB and rtcBA operons; intragenic promoters were identified in the ChIP-chip assay (see Results). Controls are the σ70-dependent, circle-junction promoter from IS492 and the empty vector, pDS12.

b β-galactosidase assays were performed in MOPS minimal medium (WT and WT+DctD250) or in nitrogen-limiting MOPS (WT-N).

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 rpoNrpoN+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 [26]. 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.
Table 2

Microarray results for known, predicted, and novel σ 54 -dependent operons and sRNA genes of S . Typhimurium

Locus taga

Gene symbolb

Function

bEBPc

WT/ΔrpoNd

Ref.e

Known σ 54 - dependent operons and sRNA genes:

   

STM0368-71

prpBCDE

Proprionate catabolism (putative)

PrpR

45

[4]

STM0830-28

glnHPQ

Glutamine high-affinity transporter

NtrC

7.1

[27]

STM2355

argT

Lysine/arginine/ornithine transport protein

NtrC

3.5

[2]

STM_R0152

glmY

GlmY sRNA

GlrR

0.9

[28]

STM_R0167

glmZ

GlmZ sRNA

GlrR

1.1

[28]

STM4007-05

glnALG

Glutamine synthetase

NtrC

48

[29]

Predicted σ 54 - dependent operons:

   

STM0462-63

glnK amtB

hypothetical protein

NtrC

3.6

[22]

STM0577-72

 

PTS (putative)

STM0571

67

[18]

STM0649.S-53

 

Hydrolase (putative)

STM0652

11

[18]

STM0665-62

gltIJKL

Glutamate/aspartate transporter

NtrC

1.8

[18, 30]

STM1285-84

yeaGH

Serine protein kinase (putative)

NtrC

2.5

[18, 30]

STM1303-07

astCABDE

Arginine/ornithine/glutamine metabolism

NtrC

2.4 f

[31, 32]

STM1690-86

pspABCDE

Phage shock proteins

PspF

17

[5]

STM2360-56

------ ubiX

Amino acid transport (putative)

STM2361

100

[18]

STM2840-41

norV ygbD

Nitric oxide reductase

NorR

16

[18, 33]

STM2843-42

hydN hypF

Hydrogenase maturation proteins

FhlA

13

[34]

STM2853-44

hycABCDEFGHI -

Hydrogenase 3

FhlA

26

[35]

STM2854-58

hypABCDE

Formate-hydrogen lyase system

FhlA

5.6

[35]

STM3521-18

- rtcBA

RNA repair system (putative)

RtcR

71

[23]

STM3568

rpoH

Heat shock sigma factor (σ32)

 

1.7

[36, 37]

STM3772-66

 

PTS (putative)

STM3773

39

[18]

STM4172

zraP

Zinc resistance-associated protein

ZraR

16

[3, 18]

STM4173-74

hydHG

Zinc resistance two-component system

ZraR

3.7

[3]

STM4244

pspG

Phage shock protein

PspF

1.4

[38]

STM4285

fdhF

Formate dehydrogenase

FhlA

29

[39]

STM4535-40.s

 

PTS (putative)

STM4534

16

[18]

Novel σ 54 - dependent operon:

   

STM2944-2937

 

CRISPR-associated genes

 

1.6 f

--

aLocus tags for genes within operons or sRNA genes are grouped by those previously shown to be σ54-dependent in Salmonella, previously predicted to be σ54-dependent, or identified in this study as encoded in a novel σ54-dependent transcript. Locus tags for operons that are not up-regulated are in bold type.

b Genes that have not been assigned a gene symbol are represented by a dash (−).

c Known or predicted bacterial enhancer-binding protein (bEBP) that activates the σ54-dependent operon.

d Signal ratio for the first gene in the operon in WT and ΔrpoN strains expressing DctD250 from pPHBP92. Operons with at least one gene with a signal ratio >3 and p-value <0.02 are considered up-regulated by RpoN; signal ratios above the 3-fold cut off are in bold type. Data for all genes in these operons can be found in Additional file1.

e References for operons shown to be σ54-dependent in Salmonella and for operons either determined to be σ54-dependent in other bacterial genera or predicted to be regulated by σ54 in Salmonella are listed.

fThe first gene in the operon was <3-fold up-regulated, but other genes in the operon were >3-fold up-regulated.

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[4], glnHPQ[27], argT[2], and glnALG[29]. Additionally, two sRNA genes, glmY and glmZ, have also been shown to have σ54-dependent promoters [28]. 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 [28]. Gopel et al. [28] 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 [29], 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, 3039]. 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 [18]. 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 [18]. 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 [40]. 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[36]. The conservation of this promoter region for rpoH in S. Typhimurium LT2 suggests that a similar complex regulatory scheme may be involved [37], 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[41]; however, our assays utilized S. Typhimurium LT2, which has a defective rpoS gene due to a transversion mutation in the start codon [42]. 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. [13] 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 [43]. The response to phage infection, which is referred to as phage shock, is regulated by σ54 and the bEBP PspF in E. coli[5]; 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 [44]. 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 [8], 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 [45]. 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. [9] and comparison analysis of the known Salmonella σ54-dependent promoters. This PSSM was applied with the Motif Locator program [46] 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.
Table 3

ChIP-chip signal ratios, PSSM scores, and predicted binding sites for ORFs enriched in the presence of σ 54

Locus taga

Gene name

Signal ratiob

Orientationc

PSSMd

Start

End

Sequence

Sites located within intergenic regions:

STM0368

prpB

11

+

20.7

417914

417931

TGGCATAGCCTTTGCTTT

STM0448

clpP

4.6

+

14.6

503028

503045

TGTCACGTATTTTGCATG

STM0462

glnK

3.2

+

20.0

520445

520462

TGGCACATCCTTTGCAAT

STM0577

 

8.3

+

17.9

636883

636866

TGGCACGCCGTTTGCCAT

STM0649.S

 

6.9

+

18.7

711945

711962

TGGCACGCCTTTTGATTA

STM0665

gltI

3.2

+

22.1

730107

730090

TGGCACGTCTATTGCTTT

STM0830

glnH

16

+

20.8

897079

897062

TGGCATGATTTTTTCATT

STM1285

yeaG

4.1

+

21.5

1363884

1363867

TGGCATGAGAGTTGCTTT

STM1303

astC

4.0

+

21.7

1382105

1382122

TGGCACGAATGCTGCAAT

STM1690

pspA

23

+

20.3

1782486

1782469

TGGCACGCAAATTGTATT

STM2355

argT

3.5

+

16.2

2466359

2466376

TGGCATAAGACCTGCATG

STM2360

 

4.8

+

23.6

2472731

2472714

TGGCATGCCTTTTGCTTT

STM_R0152

glmY

31

+

20.6

2707874

2707857

TGGCACAATTACTGCATA

STM2809

proV

9.5

-

14.6

2955839

2955822

TGGCATGAATATTGCGAG

STM2840

 

6.5

+

20.1

2985009

2985026

TGGCACACTAGCTGCAAT

STM2843

hydN

23

+

17.1

2990721

2990704

TGGCACGATTCGTGTATA

STM2853

hycA

31

+

17.9

2999639

2999622

TGGCATGGAAAATGCTTA

STM2854

hypA

71

+

22.4

2999753

2999770

TGGCATAAATATTGCTTT

STM3521

rsr

15

+

21.2

3684734

3684717

TGGCACGCTGGTTGCAAT

STM3568

rpoH

22

+

18.9

3736836

3736819

TGGCACGGTTGTTGCTCG

STM3772

dgaA

3.6

+

20.3

3972484

3972467

TGGCACAACCTTTGCTCT

STM_R0167

glmZ

15

+

19.5

4141620

4141637

TGGCACGTTATGTGCAAT

STM4007

glnA

4.2

+

19.2

4217110

4217093

TGGCACAGATTTCGCTTT

STM4172

zraP

29

+

17.4

4388217

4388234

TGGCACGGAAGATGCAAG

STM4173

hydH

4.8

+

20.1

4388385

4388402

TGGCATGATCTCTGCTTA

STM4244

pspG

39

+

19.4

4465042

4465059

TGGCATGATTTTTGTAAG

STM4285

fdhF

10

+

18.2

4527564

4527547

TGGCATAAAACATGCATA

STM4367

yjeB

3.8

+

14.1

4610407

4610424

TGGCAGATATTTTGCTTG

STM4535

 

12

+

18.3

4794881

4794898

TGGCACGCCGCTTGCTCT

Sites located within the enriched ORF:

STM0131

ftsQ

7.6

+

6.2

153598

153615

TGGAACGCGTCTTGCAGG

STM0155

 

4.1

+

9.5

182767

182784

CGGCATGGCATTTGCCAG

STM0322

proA

7.8

-

11.3

368058

368041

CGGCACAGTTTATGCAAG

STM0332

 

3.0

-

8.1

376286

376269

TGGCCAGAAATATGCTTA

STM0526

ylbA

4.3

+

9.1

588233

588216

TGGCATTAATGCTGCATC

STM0699

 

14

+

13.7

761691

761674

TGGCATCGATATTGCAAA

STM0879^

potH

5.2

+

12.1

951550

951567

TGGCAGGAGTTTTTCAAT

STM0884

ulaA

5.2

+

10.0

955545

955562

CGGCACGATTTTTTCCAT

STM0901

 

3.9

+

11.7

971761

971778

TGGCATGAAACTTGTCAC

STM0940

 

9.5

+

13.7

1018097

1018080

TGGCCTGAATCTTGCTAA

STM0961

 

7.6

-

17.7

1041686

1041669

TGGCATGAAAGCTGCTCA

STM1361

ydiM

3.6

+

11.6

1443903

1443886

TGGCATTCTTTATGCTCA

STM1390

orf242

8.9

-

12.9

1475563

1475546

TGGCATCATTATTGCCTA

STM1409

ssaJ

5.0

+

6.5

1490273

1490290

TGGCATGAAGGTTCATCG

STM1586

 

6.6

-

13.5

1672845

1672862

TGGCAAGAATATTGCCAT

STM1594

srfB

4.8

+

13.4

1681565

1681582

TGGCACACGTTTTGCGCT

STM1665

 

4.2

-

11.7

1759185

1759168

TGGCATCATTTTTTCAAG

STM1904^

yecN

3.6

+

5.9

1998988

1999005

TGGCAAACCTGTGGTATA

STM1928

otsA

5.3

+

8.1

2023398

2023381

TGGCAGGAGCGTTTTATT

STM1990

yedA

5.6

+

14.4

2072998

2073015

TGGCGCGCTTTTTGCCTT

STM2033

cbiC

4.4

-

2.2

2111221

2111238

CGGTATAAATAATGCACG

STM2115

wcaA

4.3

-

9.5

2198775

2198792

TGGCATATAAATTGAGAT

STM2181

yohJ

15

+

4.9

2277993

2278010

AGGCATTTTTCTTGCATC

STM2430

cysK

4.8

-

11.7

2544207

2544190

TGGCATCACTGTTGCAGT

STM2475

 

9.0

-

1.0

2585621

2585638

TGGCACATCAGGCAAAAG

STM2476

ypfG

3.1

+

12.7

2586874

2586857

TGGCAGGTCACCTGCAAT

STM2517

sinH

4.6

-

11.1

2650462

2650479

TGGTACGGATCTTGCCAT

STM2563

yfhG

4.7

-

6.8

2705786

2705803

CGGCGTAATTTTTGCATC

STM2939

ygcH

10

+

10.9

3080061

3080044

CGGCACAGCTCTTGCATC

STM2957

rumA

5.5

+

14.5

3105809

3105792

TGGAACGCTTTTCGCATT

STM3072

 

4.1

-

6.9

3234181

3234164

TGGCCCATTGAATGCATC

STM3302

yhbE

5.5

+

12.6

3472042

3472025

TGGCATGATGGTCGCCAG

STM3535

glgA

8.0

+

11.6

3702315

3702298

AGGCATGTTTTATGCAAA

STM3721

rfaP

13.5

+

8.3

3916283

3916300

TGGTACGTAAAATGCACG

STM3863^

 

7.5

+

11.1

4072959

4072942

TGGCGCGATTATTGCCAG

STM3919

wzzE

4.2

+

11.0

4128295

4128312

TGGCCTGCTATTTGCCCT

STM3924

wecD

22

+

11.8

4133232

4133249

TGGCGCGGAAATTGCACA

STM4013.S

 

3.6

-

13.6

4222708

4222725

TGGCATAAAACCTGAAAA

STM4226

yjbA

6.4

-

4.2

4446318

4446301

AGGCGCGAATAATGCATC

STM4290

proP

13

+

10.1

4532022

4532039

TGGCCTGATTTTTGCAGG

STM4572

stjB

8.3

-

8.2

4826908

4826925

TGGCGTGGCGATTTCAAT

aLoci listed in bold are known or predicted σ54-dependent promoters.

bRatio of signals between WT and ΔrpoN cells.

cOrientation of the predicted binding site with respect to the listed ORF. (+) binding site is in same direction as ORF; (−) binding site is in opposite direction as ORF.

dPSSM score for the best predicted binding site within 1 kb of enriched ORF using the position-specific scoring matrix derived from the sequences in Additional file 2. As a reference to interpret the PSSM scores, the S. Typhimurium LT2 chromosome contains 3 sites with PSSM scores ≥22.0, 21 sites with PSSM scores ≥18.0, 61 sites with PSSM scores ≥14.0, and 401 sites with PSSM scores ≥10.0.

^The predicted binding site is a potential promoter for a neighboring gene based on its orientation and location within 250 bp of the 5’ end of a neighboring gene or a long intergenic region (>100 bp) that may encode a sRNA.

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.
Figure 2

Binding sites predicted by ChIP-chip analysis. A) Location of the predicted binding sites for the 70 ORFs enriched by α-σ54 pulldown. Outer bars represent further breakdown by location and orientation of the binding site relative to the enriched ORF, as diagrammed in (B). A (+) indicates that the binding site is in the same orientation as the ORF while (−) indicates that the binding site is in the opposite orientation as the enriched ORF.

Consensus sequences were generated using WebLogo [47] 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 [48]. 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 [49]. 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.
Figure 3

Alignment of σ 54 binding sites. Weblogos show the consensus sequence for A) 27 known/predicted promoter sequences used to generate the position-specific scoring matrix B) 29 predicted intergenic binding sites for ORFs enriched in ChIP-chip analysis or C) 41 predicted intragenic binding sites from within ORFs enriched in ChIP-chip analysis. Weblogos were generated using the online program available at http://weblogo.berkeley.edu/.

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 [19]. 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 [20]. Binding sites located near a functional σ54 promoter may serve to accelerate the search for the promoter by Eσ54 sliding from the secondary sites [50]; 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 [51]. 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 [52], 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 [53], thus improving the chances of cross-linking Eσ54 at the promoter sequence [54].

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].
Figure 4

Promoter location, orientation, and activity for selected σ 54 binding sites. The ratio of β-galactosidase activity (Miller Units) in WT+DctD250 vs. ΔrpoN+DctD250 cells is shown for (A) known (light blue bars) and predicted (dark blue bars) σ54-dependent promoters, and (B) potential intragenic promoter sequences (red bars) in the promoter reporter vectors, pDS11 or pDS12 (black bars). Double asterisks denote significant increase in β-galactosidase activity in WT+DctD250 versus ΔrpoN+DctD250 (p-value <0.02). Circled numbers below locus tags indicate orientation of the potential promoter sequence, as illustrated in (C). Orientation of potential intragenic promoter sequence is: 1) same as ORF and >300 bp from 3’ end; 2) same as ORF and <300 bp from 3’ end of a convergent downstream gene; 3) opposite of ORF and >300 bp from 5’ end; 4) opposite of ORF and <300 bp from the 3’ end of an upstream gene; and 5) opposite of ORF and <300 bp from 5’ end of gene, but >300 bp from the 3’ end of an upstream gene.

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 [55]).

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.
Figure 5

Comparison of positive results from characterization of the σ 54 regulon for S . Typhimurium LT2. + Positive results are promoter sequences that were up-regulated >3-fold, or displayed a significant increase in β-galactosidase activity in WT+DctD250 compared to ΔrpoN+DctD250 in DNA microarray, and promoter-lacZ fusion assays, respectively or enriched >3-fold in WT compared to ΔrpoN cells in ChIP-chip assays. Regions of overlap indicate promoters that were positive in multiple experiments.

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 [42]. Changes in the level of expression of one sigma factor can alter the expression of genes that are expressed by different sigma factors [56]; for example, it has been shown that deletion of rpoN alters expression of σS-dependent promoters in E. coli[57]. 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 [1315, 5860]. Only in Vibrio cholera 037 strain V52 have both global transcripts and binding sites been characterized experimentally [14]. 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. [13] 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 [14] 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 [8]); 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 [61]. 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 [62] and there is extensive conservation of amino acid sequence in Region I for σ54 from phylogenetically diverse bacteria [63]. 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.

Conclusions

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.

Methods

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-] [64]. An isogenic derivative, TRH134, has a deletion in rpoN (ntrA) from codons 8 through 455, rendering it auxotrophic for glutamine [65]. S. Typhimurium strains were cultured in either nutrient broth (NB; Difco Laboratories), MOPS minimal media [66], or nitrogen-limiting MOPS [67]. 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.

Plasmids

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 [17] 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 [25] 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/lacI q , 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 [68].

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 [17], 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 [69]. Labeled cDNA was hybridized to DNA microarrays containing complete open reading frames (ORFs) from S. Typhimurium LT2 printed in triplicate [70]. 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 [70]. 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[73] 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.

ChIP-chip assays

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 [46]. 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.

β-Galactosidase assays

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 [74] 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))] [74]. 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.

Abbreviations

Amp: 

Ampicillin

bEBP: 

Bacterial enhancer-binding protein

ChIP: 

Chromatin immunoprecipitation

Eσ: 

RNA polymerase holoenzyme

LB: 

Luria-bertani media

LM-PCR: 

Ligation-mediated PCR

NB: 

Nutrient broth

RNAP: 

RNA polymerase

Spc: 

Spectinomycin

Str: 

Streptomycin

UAS: 

Upstream activation sequence.

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Department of Microbiology, University of Georgia
(2)
US Department of Agriculture, Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Richard B. Russell Research Center, Agricultural Research Service
(3)
Department of Microbiology and Molecular Genetics, University of California
(4)
Institute of Bioinformatics, University of Georgia

References

  1. Gruber TM, Gross CA: Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol. 2003, 57: 441-466. 10.1146/annurev.micro.57.030502.090913.View ArticlePubMedGoogle Scholar
  2. Ferro-Luzzi Ames G, Nikaido K: Nitrogen regulation in Salmonella typhimurium. Identification of an NtrC protein-binding site and definition of a consensus binding sequence. EMBO J. 1985, 4 (2): 539-547.PubMed CentralPubMedGoogle Scholar
  3. Leonhartsberger S, Huber A, Lottspeich F, Bock A: The hydH/G Genes from Escherichia coli code for a zinc and lead responsive two-component regulatory system. J Mol Biol. 2001, 307 (1): 93-105. 10.1006/jmbi.2000.4451.View ArticlePubMedGoogle Scholar
  4. Palacios S, Escalante-Semerena JC: prpR, ntrA, and ihf functions are required for expression of the prpBCDE operon, encoding enzymes that catabolize propionate in Salmonella enterica serovar Typhimurium LT2. J Bacteriol. 2000, 182 (4): 905-910. 10.1128/JB.182.4.905-910.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Weiner L, Brissette JL, Model P: Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma54 and modulated by positive and negative feedback mechanisms. Genes Dev. 1991, 5 (10): 1912-1923. 10.1101/gad.5.10.1912.View ArticlePubMedGoogle Scholar
  6. Niehus E, Gressmann H, Ye F, Schlapbach R, Dehio M, Dehio C, Stack A, Meyer TF, Suerbaum S, Josenhans C: Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori. Mol Microbiol. 2004, 52 (4): 947-961. 10.1111/j.1365-2958.2004.04006.x.View ArticlePubMedGoogle Scholar
  7. Wigneshweraraj S, Bose D, Burrows PC, Joly N, Schumacher J, Rappas M, Pape T, Zhang X, Stockley P, Severinov K: Modus operandi of the bacterial RNA polymerase containing the σ54 promoter-specificity factor. Mol Microbiol. 2008, 68 (3): 538-546. 10.1111/j.1365-2958.2008.06181.x.View ArticlePubMedGoogle Scholar
  8. Bush M, Dixon R: The role of bacterial enhancer binding proteins as specialized activators of sigma54-dependent transcription. Microbiol Mol Biol Rev. 2012, 76 (3): 497-529. 10.1128/MMBR.00006-12.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Barrios H, Valderrama B, Morett E: Compilation and analysis of sigma(54)-dependent promoter sequences. Nucleic Acids Res. 1999, 27 (22): 4305-4313. 10.1093/nar/27.22.4305.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Ghosh T, Bose D, Zhang X: Mechanisms for activating bacterial RNA polymerase. FEMS Microbiol Rev. 2010, 34 (5): 611-627.View ArticlePubMedGoogle Scholar
  11. Belitsky BR, Sonenshein AL: An enhancer element located downstream of the major glutamate dehydrogenase gene of Bacillus subtilis. Proc Natl Acad Sci USA. 1999, 96 (18): 10290-10295. 10.1073/pnas.96.18.10290.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Reitzer LJ, Magasanik B: Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell. 1986, 45 (6): 785-792. 10.1016/0092-8674(86)90553-2.View ArticlePubMedGoogle Scholar
  13. Zhao K, Liu M, Burgess RR: Promoter and regulon analysis of nitrogen assimilation factor, sigma54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis. Nucleic Acids Res. 2010, 38 (4): 1273-1283. 10.1093/nar/gkp1123.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Dong TG, Mekalanos JJ: Characterization of the RpoN regulon reveals differential regulation of T6SS and new flagellar operons in Vibrio cholerae O37 strain V52. Nucleic Acids Res. 2012, 40 (16): 7766-7775. 10.1093/nar/gks567.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Leang C, Krushkal J, Ueki T, Puljic M, Sun J, Juarez K, Nunez C, Reguera G, DiDonato R, Postier B: Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens. BMC Genomics. 2009, 10: 331-10.1186/1471-2164-10-331.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Brahmachary P, Dashti MG, Olson JW, Hoover TR: Helicobacter pylori FlgR is an enhancer-independent activator of σ54-RNA polymerase holoenzyme. J Bacteriol. 2004, 186 (14): 4535-4542. 10.1128/JB.186.14.4535-4542.2004.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Xu H, Gu B, Nixon BT, Hoover TR: Purification and characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a sigma54-dependent transcriptional activator. J Bacteriol. 2004, 186 (11): 3499-3507. 10.1128/JB.186.11.3499-3507.2004.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Studholme DJ: Enhancer-dependent transcription in Salmonella enterica Typhimurium: new members of the sigmaN regulon inferred from protein sequence homology and predicted promoter sites. J Mol Microbiol Biotechnol. 2002, 4 (4): 367-374.PubMedGoogle Scholar
  19. Georg J, Hess WR: cis-antisense RNA, another level of gene regulation in bacteria. Microbiol Mol Biol Rev. 2011, 75 (2): 286-300. 10.1128/MMBR.00032-10.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Gottesman S, Storz G: Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harbor perspectives in biology. 2011, 3: 12-View ArticleGoogle Scholar
  21. Kroger C, Dillon SC, Cameron AD, Papenfort K, Sivasankaran SK, Hokamp K, Chao Y, Sittka A, Hebrard M, Handler K: The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proc Natl Acad Sci USA. 2012, 109 (20): E1277-1286. 10.1073/pnas.1201061109.PubMed CentralView ArticlePubMedGoogle Scholar
  22. van Heeswijk WC, Hoving S, Molenaar D, Stegeman B, Kahn D, Westerhoff HV: An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. Mol Microbiol. 1996, 21 (1): 133-146. 10.1046/j.1365-2958.1996.6281349.x.View ArticlePubMedGoogle Scholar
  23. Genschik P, Drabikowski K, Filipowicz W: Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its sigma54-regulated operon. J Biol Chem. 1998, 273 (39): 25516-25526. 10.1074/jbc.273.39.25516.View ArticlePubMedGoogle Scholar
  24. Huala E, Stigter J, Ausubel FM: The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol. 1992, 174 (4): 1428-1431.PubMed CentralPubMedGoogle Scholar
  25. Perkins-Balding D, Duval-Valentin G, Glasgow AC: Excision of IS492 requires flanking target sequences and results in circle formation in Pseudoalteromonas atlantica. J Bacteriol. 1999, 181 (16): 4937-4948.PubMed CentralPubMedGoogle Scholar
  26. Porwollik S, Frye J, Florea LD, Blackmer F, McClelland M: A non-redundant microarray of genes for two related bacteria. Nucleic Acids Res. 2003, 31 (7): 1869-1876. 10.1093/nar/gkg298.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Klose KE, Mekalanos JJ: Simultaneous prevention of glutamine synthesis and high-affinity transport attenuates Salmonella typhimurium virulence. Infect Immun. 1997, 65 (2): 587-596.PubMed CentralPubMedGoogle Scholar
  28. Gopel Y, Luttmann D, Heroven AK, Reichenbach B, Dersch P, Gorke B: Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae. Nucleic Acids Res. 2011, 39 (4): 1294-1309. 10.1093/nar/gkq986.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Hirschman J, Wong PK, Sei K, Keener J, Kustu S: Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci USA. 1985, 82 (22): 7525-7529. 10.1073/pnas.82.22.7525.PubMed CentralView ArticlePubMedGoogle Scholar
  30. Zimmer DP, Soupene E, Lee HL, Wendisch VF, Khodursky AB, Peter BJ, Bender RA, Kustu S: Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation. Proc Natl Acad Sci USA. 2000, 97 (26): 14674-14679. 10.1073/pnas.97.26.14674.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Kiupakis AK, Reitzer L: ArgR-independent induction and ArgR-dependent superinduction of the astCADBE operon in Escherichia coli. J Bacteriol. 2002, 184 (11): 2940-2950. 10.1128/JB.184.11.2940-2950.2002.PubMed CentralView ArticlePubMedGoogle Scholar
  32. Riordan JT, Tietjen JA, Walsh CW, Gustafson JE, Whittam TS: Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157: H7. Microbiology. 2010, 156 (Pt 3): 719-730.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Gardner AM, Gessner CR, Gardner PR: Regulation of the nitric oxide reduction operon (norRVW) in Escherichia coli. Role of NorR and σ54 in the nitric oxide stress response. J Biol Chem. 2003, 278 (12): 10081-10086. 10.1074/jbc.M212462200.View ArticlePubMedGoogle Scholar
  34. Maier T, Binder U, Bock A: Analysis of the hydA locus of Escherichia coli: two genes (hydN and hypF) involved in formate and hydrogen metabolism. Arch Microbiol. 1996, 165 (5): 333-341. 10.1007/s002030050335.View ArticlePubMedGoogle Scholar
  35. Lutz S, Bohm R, Beier A, Bock A: Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli. Mol Microbiol. 1990, 4 (1): 13-20. 10.1111/j.1365-2958.1990.tb02010.x.View ArticlePubMedGoogle Scholar
  36. Janaszak A, Majczak W, Nadratowska B, Szalewska-Palasz A, Konopa G, Taylor A: A sigma54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene. Microbiology. 2007, 153 (Pt 1): 111-123.View ArticlePubMedGoogle Scholar
  37. Ramirez-Santos J, Collado-Vides J, Garcia-Varela M, Gomez-Eichelmann MC: Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria. Nucleic Acids Res. 2001, 29 (2): 380-386. 10.1093/nar/29.2.380.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Lloyd LJ, Jones SE, Jovanovic G, Gyaneshwar P, Rolfe MD, Thompson A, Hinton JC, Buck M: Identification of a new member of the phage shock protein response in Escherichia coli, the phage shock protein G (PspG). J Biol Chem. 2004, 279 (53): 55707-55714. 10.1074/jbc.M408994200.View ArticlePubMedGoogle Scholar
  39. Birkmann A, Sawers RG, Bock A: Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli. Mol Gen Genet. 1987, 210 (3): 535-542. 10.1007/BF00327209.View ArticlePubMedGoogle Scholar
  40. Miller KA, Phillips RS, Mrazek J, Hoover TR: Salmonella utilizes D-glucosaminate via a mannose family phosphotransferase system permease and associated enzymes. J Bacteriol. 2013, 195 (18): 4057-4066. 10.1128/JB.00290-13.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Ibanez-Ruiz M, Robbe-Saule V, Hermant D, Labrude S, Norel F: Identification of RpoS (sigmaS)-regulated genes in Salmonella enterica serovar typhimurium. J Bacteriol. 2000, 182 (20): 5749-5756. 10.1128/JB.182.20.5749-5756.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Swords WE, Cannon BM, Benjamin WH: Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene. Infect Immun. 1997, 65 (6): 2451-2453.PubMed CentralPubMedGoogle Scholar
  43. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P: CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007, 315 (5819): 1709-1712. 10.1126/science.1138140.View ArticlePubMedGoogle Scholar
  44. Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA: Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure. 2009, 17 (6): 904-912. 10.1016/j.str.2009.03.019.View ArticlePubMedGoogle Scholar
  45. Buck M, Cannon W: Specific binding of the transcription factor σ54 to promoter DNA. Nature. 1992, 358 (6385): 422-424. 10.1038/358422a0.View ArticlePubMedGoogle Scholar
  46. Mrazek J: Finding sequence motifs in prokaryotic genomes–a brief practical guide for a microbiologist. Brief Bioinform. 2009, 10 (5): 525-536. 10.1093/bib/bbp032.View ArticlePubMedGoogle Scholar
  47. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res. 2004, 14 (6): 1188-1190. 10.1101/gr.849004.PubMed CentralView ArticlePubMedGoogle Scholar
  48. Doucleff M, Pelton JG, Lee PS, Nixon BT, Wemmer DE: Structural basis of DNA recognition by the alternative sigma-factor, σ54. J Mol Biol. 2007, 369 (4): 1070-1078. 10.1016/j.jmb.2007.04.019.PubMed CentralView ArticlePubMedGoogle Scholar
  49. Cannon W, Wigneshweraraj SR, Buck M: Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA. Nucleic Acids Res. 2002, 30 (4): 886-893. 10.1093/nar/30.4.886.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Li GW, Elf J: Single molecule approaches to transcription factor kinetics in living cells. FEBS Lett. 2009, 583 (24): 3979-3983. 10.1016/j.febslet.2009.11.035.View ArticlePubMedGoogle Scholar
  51. Johansson LU, Solera D, Bernardo LM, Moscoso JA, Shingler V: sigma54-RNA polymerase controls sigma70-dependent transcription from a non-overlapping divergent promoter. Mol Microbiol. 2008, 70 (3): 709-723. 10.1111/j.1365-2958.2008.06440.x.View ArticlePubMedGoogle Scholar
  52. Friedman LJ, Gelles J: Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation. Cell. 2012, 148 (4): 679-689. 10.1016/j.cell.2012.01.018.PubMed CentralView ArticlePubMedGoogle Scholar
  53. Feklistov A, Mekler V, Jiang Q, Westblade LF, Irschik H, Jansen R, Mustaev A, Darst SA, Ebright RH: Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center. Proc Natl Acad Sci USA. 2008, 105 (39): 14820-14825. 10.1073/pnas.0802822105.PubMed CentralView ArticlePubMedGoogle Scholar
  54. Raffaelle M, Kanin EI, Vogt J, Burgess RR, Ansari AZ: Holoenzyme switching and stochastic release of sigma factors from RNA polymerase in vivo. Mol Cell. 2005, 20 (3): 357-366. 10.1016/j.molcel.2005.10.011.View ArticlePubMedGoogle Scholar
  55. Maeda H, Fujita N, Ishihama A: Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase. Nucleic Acids Res. 2000, 28 (18): 3497-3503. 10.1093/nar/28.18.3497.PubMed CentralView ArticlePubMedGoogle Scholar
  56. Grigorova IL, Phleger NJ, Mutalik VK, Gross CA: Insights into transcriptional regulation and sigma competition from an equilibrium model of RNA polymerase binding to DNA. Proc Natl Acad Sci USA. 2006, 103 (14): 5332-5337. 10.1073/pnas.0600828103.PubMed CentralView ArticlePubMedGoogle Scholar
  57. Dong T, Yu R, Schellhorn H: Antagonistic regulation of motility and transcriptome expression by RpoN and RpoS in Escherichia coli. Mol Microbiol. 2011, 79 (2): 375-386. 10.1111/j.1365-2958.2010.07449.x.View ArticlePubMedGoogle Scholar
  58. Chaudhuri RR, Yu L, Kanji A, Perkins TT, Gardner PP, Choudhary J, Maskell DJ, Grant AJ: Quantitative RNA-seq analysis of the Campylobacter jejuni transcriptome. Microbiology. 2011, 157 (Pt 10): 2922-2932.PubMed CentralView ArticlePubMedGoogle Scholar
  59. Damron FH, Owings JP, Okkotsu Y, Varga JJ, Schurr JR, Goldberg JB, Schurr MJ, Yu HD: Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN. J Bacteriol. 2012, 194 (6): 1317-1330. 10.1128/JB.06105-11.PubMed CentralView ArticlePubMedGoogle Scholar
  60. Dasgupta N, Wolfgang MC, Goodman AL, Arora SK, Jyot J, Lory S, Ramphal R: A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol Microbiol. 2003, 50 (3): 809-824. 10.1046/j.1365-2958.2003.03740.x.View ArticlePubMedGoogle Scholar
  61. Francke C, Groot Kormelink T, Hagemeijer Y, Overmars L, Sluijter V, Moezelaar R, Siezen RJ: Comparative analyses imply that the enigmatic Sigma factor 54 is a central controller of the bacterial exterior. BMC Genomics. 2011, 12: 385-10.1186/1471-2164-12-385.PubMed CentralView ArticlePubMedGoogle Scholar
  62. Xiao Y, Wigneshweraraj SR, Weinzierl R, Wang YP, Buck M: Construction and functional analyses of a comprehensive sigma54 site-directed mutant library using alanine-cysteine mutagenesis. Nucleic Acids Res. 2009, 37 (13): 4482-4497. 10.1093/nar/gkp419.PubMed CentralView ArticlePubMedGoogle Scholar
  63. Buck M, Gallegos MT, Studholme DJ, Guo Y, Gralla JD: The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor. J Bacteriol. 2000, 182 (15): 4129-4136. 10.1128/JB.182.15.4129-4136.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  64. Grana D, Youderian P, Susskind MM: Mutations that improve the ant promoter of Salmonella phage P22. Genetics. 1985, 110 (1): 1-16.PubMed CentralPubMedGoogle Scholar
  65. Kelly MT, Hoover TR: Mutant forms of Salmonella typhimurium sigma54 defective in transcription initiation but not promoter binding activity. J Bacteriol. 1999, 181 (11): 3351-3357.PubMed CentralPubMedGoogle Scholar
  66. Maloy S, Stewart VJ, Taylor RK: Genetic Analysis of Pathogenic Bacteria: A Laboratory Manual. 1996, Plainview, NY: Cold Spring Harbor Laboratory PressGoogle Scholar
  67. Kustu SG, McFarland NC, Hui SP, Esmon B, Ames GF: Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. J Bacteriol. 1979, 138 (1): 218-234.PubMed CentralPubMedGoogle Scholar
  68. Hughes KT, Youderian P, Simon MI: Phase variation in Salmonella: analysis of Hin recombinase and hix recombination site interaction in vivo. Genes Dev. 1988, 2 (8): 937-948. 10.1101/gad.2.8.937.View ArticlePubMedGoogle Scholar
  69. Wang Q, Frye JG, McClelland M, Harshey RM: Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes. Mol Microbiol. 2004, 52 (1): 169-187. 10.1111/j.1365-2958.2003.03977.x.View ArticlePubMedGoogle Scholar
  70. Porwollik S, McClelland M: Lateral gene transfer in Salmonella. Microbes Infect. 2003, 5 (11): 977-989. 10.1016/S1286-4579(03)00186-2.View ArticlePubMedGoogle Scholar
  71. Xia XQ, McClelland M, Porwollik S, Song W, Cong X, Wang Y: WebArrayDB: cross-platform microarray data analysis and public data repository. Bioinformatics. 2009, 25 (18): 2425-2429. 10.1093/bioinformatics/btp430.PubMed CentralView ArticlePubMedGoogle Scholar
  72. Xia X, McClelland M, Wang Y: WebArray: an online platform for microarray data analysis. BMC Bioinforma. 2005, 6: 306-10.1186/1471-2105-6-306.View ArticleGoogle Scholar
  73. Kelly MT, Hoover TR: The amino terminus of Salmonella enterica serovar Typhimurium sigma54 is required for interactions with an enhancer-binding protein and binding to fork junction DNA. J Bacteriol. 2000, 182 (2): 513-517. 10.1128/JB.182.2.513-517.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  74. Miller JH: A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. 1992, Plainview, NY: Cold Spring Harbor PressGoogle Scholar

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