Bacterial strains and reporter gene constructs

Salmonella enterica serovar Typhimurium strain SL1344 and mutant derivatives were used for all experiments. Detailed descriptions of strains used in this study are provided in Table S1. Deletion mutants were constructed by the method of Datsenko and Wanner [22] using the recombineering primers listed in Table S2. Mutations were transduced into a fresh SL1344 background by bacteriophage P22 generalized transduction [23] and were confirmed by PCR and DNA sequencing. The pCP20 plasmid, which encodes the FLP recombinase, was used to remove the kanamycin resistance cassette from the dusB-fis-gfp^TCD::kan^R construct in the S. Typhimurium chromosome as described in [22]. After curing cells of pCP20, loss of antibiotic resistance was confirmed by screening colonies for kanamycin sensitivity using LB-plates containing kanamycin at 50 μg/ml. Construction of SL1344 Δcrp was confirmed phenotypically by sugar fermentations tests using peptone broth containing a sugar or sugar alcohol at 0.5% final concentration and bromothymol blue as an indicator. As expected SL1344 Δcrp was unable to ferment mannitol, sorbitol or maltose but retained its ability to ferment glucose.

Promoter truncates were generated by inverse PCR using the primers listed in Table S2 and pZepfisS as template [13]. PCR amplicons were phosphorylated by T4 polynucleotide kinase and then circularised by ligation with T4 DNA ligase, followed by transformation into chemically-competent E. coli DH5α. PCR primers targeting the fis promoter region included a restriction enzyme site to facilitate confirmation of correct ligation products.

Culture growth

Well-aerated conditions were achieved by using 10 ml of LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) in a 250 ml glass conical flask, whereas non-aerated conditions used the same culture volume in a glass tube with an interior diameter 14 mm, as in [13]. All containers were loosely capped to allow air flow, and shaken at 200 RPM at 37 °C. Cultures were started by diluting cells 1/100 from well-aerated overnight cultures and grown for 22 hours to ensure that they had reached steady-state stationary phase before sampling. Anaerobic shock was administered by transferring 15 ml of well-aerated exponentially-growing cell culture into a 15 ml centrifuge tube, capping tightly, and incubating without agitation for 30 min at 37 °C. When required, antibiotics were used at the following final concentrations: carbenicillin 100 µg/ml, chloramphenicol 20 µg/ml, and kanamycin 50 µg/ml.

Flow cytometry and quantitative PCR

GFP levels in cells expressing fis-gfp^TCD and Pfis-gfp+ were quantified using flow cytometry as follows: 2-30 µl of culture was fixed in 700 µl of freshly prepared phosphate buffered saline containing 2% formaldehyde. Fixed samples were stored overnight in the dark at 4 °C. The median fluorescence of 20,000 cells/sample was measured on a Dako CyAn ADP flow cytometer (PMT voltage 800-875 V). To quantify transcript abundance, RNA was isolated using TRIzol as described earlier [24]. Quantitative PCR was conducted as in [25] using primers listed in Table S2.

DNA supercoiling analysis

Plasmids were isolated from cultures using the HiYield Plasmid Mini Kit (RBC Bioscience). All electrophoresis was conducted in 27-cm-long 1% agarose gels with 2x Tris Borate EDTA (TBE) as gel and running buffer. Approximately 1 µg of plasmid DNA (8–15 µl) was loaded on a gel using 4 µl of loading buffer (80% glycerol, 0.5 mg/ml bromophenol blue). The gel and buffer contained 2.5 µg/ml chloroquine, and electrophoresis was performed at 3 V/cm for 16 hours. To remove the chloroquine after electrophoresis, gels were washed by gentle rocking in large volumes of tap water for at least 3 hours; the wash water was replaced every 20–30 minutes. After washing, gels were stained by gentle rocking in water containing ethidium bromide (1 µg/ml) for at least 1 hour, then washed briefly in water, and plasmid topoisomers were visualized with UV light. Analyses of topoisomer distributions were conducted as in [26].

WebSIDD [27] was used to predict how DNA supercoiling affects stability of the S. Typhimurium and E. coli fis promoter regions. WebSIDD uses a default 5 kbp window that slides by 500 bp. Thus the stability of each base pair, G(x), is the average of 10 calculations, where the influence of proximal bases is weighted according to how close the base pair is to the center of the sliding window.

5′ RACE and western blot analysis

5′ RACE was conducted as in [28] using the primer Pfis.expTSS.RT.Rev to reverse transcribe dusB-fis transcripts. PCR products were cloned into the linearized vector pJET (Fermentas) and transformed into E. coli strain XL-1 and at least 5 clones were DNA sequenced. Western blot analysis was conducted as in [25] using the E. coli σ^S (RpoS) monoclonal antibody (Neoclone) diluted 1:5,000.

Comparison of S. Typhimurium and E. coli fis promoter regions

The regulation of fis expression has been studied primarily in E. coli. As a first step to identify the regulatory elements that control S. Typhimurium fis expression, the S. Typhimurium fis region was aligned with the same region in E. coli. The bicistronic dusB-fis operon encodes FIS in both species. Because dusB has attracted very little research attention, the upstream promoter region (Pfis) bears the fis name. The dusB and fis open reading frames have 92% and 98% nucleotide identity, respectively, between E. coli and S. Typhimurium, indicating that very little change has occurred in these genes since divergence of the Escherichia and Salmonella genera. As has been noted previously, the region containing the primary transcription start site is identical between E. coli and S. Typhimurium [20], but there is significant sequence divergence upstream of position −49 (Figure 1). In E. coli there is an intergenic space of 296 bp between the dusB-fis transcription start site and the stop codon of the upstream gene, prmA. In S. Typhimurium, the prmA stop codon is 628 bp upstream, suggesting that >300 bp of non-coding DNA was inserted upstream of the ancestral promoter region. Alignment of DNA sequence from other Enterobacteriaceae species confirms that the ancestral promoter region was likely around 300 bp (not shown), making Salmonella unusual in the Enterobacteriaceae family.

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Figure 1. Gapless sequence alignment of the E. coli and S. Typhimurium fis promoter regions.

CRP and FIS (grey boxes) and IHF (underline) binding sites identified in E. coli are shown [5,6,40]. Promoter elements, transcription start sites and dusB translation initiation codons are boxed. The angled arrows show the locations of Pfis-1 (both species) and Pfis-2 (S. Typhimurium). The locations of promoter truncations in S. Typhimurium are indicated by black triangles.

https://doi.org/10.1371/journal.pone.0084382.g001

To test whether the full-length intergenic region contributes to regulation, the gfp^TCD reporter gene was fused to the S. Typhimurium dusB-fis operon in the native chromosomal location. Because FIS is autoregulatory, we avoided creating a fis null mutant by inserting gfp^TCD downstream of the fis open reading frame, but upstream of a predicted transcriptional terminator. The kanamycin resistance marker linked to gfp^TCD was subsequently excised to prevent the kan promoter from influencing fis expression. This chromosome-based reporter differs from the study by Ó Cróinín and Dorman that first identified sustained fis expression [13]. Ó Cróinín and Dorman used a plasmid-based reporter gene fusion containing the fis promoter region up to -298 bp, thus omitting the 330 bp of upstream non-coding sequence.

In stationary phase cultures the dusB-fis-gfp^TCD construct demonstrated elevated expression in non-aerated conditions compared to aerated conditions (Figure 2A). This modest two-fold increase in expression corresponds to a dramatic rise in FIS protein level, which causes S. Typhimurium to become more invasive in a fis-dependent manner [13]. Overall, the dusB-fis-gfp^TCD chromosomal fusion exhibited the classic expression pattern of strong induction in exponential growth in both well-aerated and non-aerated conditions (Figure 2C), followed by a rapid decrease in expression as growth slowed. Two-fold higher fluorescence was sustained from 3 to 22 hours of growth in non-aerated compared to aerated conditions (Figure 2A & C). The two-fold difference we observed with the dusB-fis-gfp^TCD construct was less than the increase seen previously in the plasmid-based reporter system [13], and quantitative PCR confirmed that our chromosomal construct is an accurate reporter of native fis gene expression (Figure S1). The reduced intensity of sustained expression in the chromosome gfp fusion compared to the plasmid-borne reporter indicates that the plasmid system is a reliable but exaggerated reporter of sustained fis expression in non-aerated conditions.

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Figure 2. Effects of transcription factor mutations on fis expression in aerated and non-aerated conditions.

A) Column graph comparing dusB-fis::gfp^TCD expression at 22 hours. All data are expressed relative to wild type at 22 hours in well-aerated conditions (dashed line). B) Average and standard deviation of four replicate culture densities after 22 hours. C-F) Time courses of dusB-fis::gfp^TCD expression in S. Typhimurium wild type and Δfnr (C), ΔarcA (D), ΔrpoS (E), and ΔarcA/ΔrpoS (F) mutants. The blue arrow in D indicates the increased sustained expression observed in the ΔarcA mutant after 22 hours in non-aerated conditions. A, C-F) Mean and standard deviation of GFP fluorescence from three or four biological replicates are plotted in arbitrary units. All data are expressed relative to wild type levels at time point 0 in well-aerated conditions. The same wild type data are presented in panels C-F. G) Growth dynamics of strains in aerated and non-aerated conditions. Smoothed curves were generated by GraphPad Prism 5.0d from the average of four or more replicate growth curves.

https://doi.org/10.1371/journal.pone.0084382.g002

ArcA is a repressor of S. Typhimurium fis

The redox sensors ArcAB and FNR are transcriptional activators in low oxygen conditions, making them prime candidates for activating sustained fis expression. Deletion of fnr had no detectable effect on fis expression or on cell growth (Figure 2B, C & G). Surprisingly, deletion of arcA caused an increase in fis expression in non-aerated stationary phase (Figure 2A & D), implicating arcA as a repressor of sustained fis expression. We found that although the ΔarcA mutant had a slightly prolonged lag phase in fresh medium, its growth rate and final cell densities were very similar to wild type cells in both aerated and non-aerated conditions (Figure 2B & G). Thus, the effect of the ΔarcA mutation on fis expression is not due to altered growth in non-aerated conditions.

In E. coli ArcA is a known repressor of rpoS transcription [29], and RpoS has been proposed to repress fis expression in S. Typhimurium [13], thus an S. Typhimurium ΔarcA mutant would be predicted to have reduced fis expression due to up-regulation of rpoS. This led us to test whether ArcA may act through rpoS in S. Typhimurium, perhaps by up-regulating rpoS expression as opposed to repressing it as in E. coli. First though we were surprised to discover that the chromosomal dusB-fis-gfp^TCD fusion was not up-regulated in the ΔrpoS mutant (Figure 2E), which contrasts with the previously observed strong activating effect of the ΔrpoS mutation on the plasmid-based Pfis::gfp fusion [13]. Quantitative PCR confirmed that the dusB-fis-gfp^TCD fusion is a reliable reporter of fis expression in non-aerated conditions (Figure S1). The ΔrpoS mutant showed an extended lag phase, but unlike the ΔarcA mutant it did not achieve wild type densities in aerated culture after 22 hours; instead it grew slightly better than wild type in non-aerated conditions (Figure 2B & G). Neither of these minor growth phenotypes had a detectable effect on fis expression.

To further address whether ArcA may act indirectly on fis through RpoS function, we constructed a ΔarcA/ΔrpoS double mutant. In aerated and non-aerated growth conditions, the ΔarcA/ΔrpoS double mutant showed consistently reduced fis expression during exponential growth (Figure 2F), unlike either single mutant. Finding an exacerbating effect of combining the two mutations suggests that ArcA acts separately from RpoS to influence fis expression in exponential growth conditions. During stationary phase in non-aerated conditions, the ΔrpoS mutation nullified the effect of the ΔarcA mutation of enhancing sustained fis expression (Figure 2A & F), implicating RpoS as a mediator of ArcA’s effect on fis expression.

The minimal fis promoter is deficient in sustained expression

Because neither FNR nor ArcA was required for sustained fis expression in non-aerated conditions, we created a series of fis promoter (Pfis) truncates to identify promoter regions required for sustained expression. Three truncates were constructed in a plasmid-based gfp reporter system. The amplification of fis expression profiles by the plasmid system provided two experimental advantages: first, the ability to create truncated promoters, and second, improved sensitivity to detect subtle changes in gene expression. Unfortunately, we were unable to clone the full intergenic region from S. Typhimurium, suggesting that an unidentified element upstream of position −300 is toxic to cells when present in multicopy. The construct Pfis(−298) contained a promoter region identical in length to the E. coli intergenic region (Figure 3A), which is the same S. Typhimurium promoter region used previously to study sustained expression [13]. The medium length construct Pfis(−198) removed sequence that in E. coli contributes to regulation (Figure 1). The shortest construct, Pfis(−49), preserved only the region of identity between S. Typhimurium and E. coli.

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Figure 3. Defining functional regions and transcription factor input at the fis promoter.

A) Schematic of the E. coli and S. Typhimurium intergenic regions. The locations of truncations in S. Typhimurium are indicated. B) Time courses of expression of plasmid-borne Pfis::gfp promoter truncates in aerated and non-aerated S. Typhimurium cultures. C and D) Expression of promoter truncates in S. Typhimurium transcription factor mutant backgrounds at 22 hours. In B-D, mean and standard deviation of GFP fluorescence from three or more biological replicates are plotted in arbitrary units that are reported relative to the chromosomal dusB-fis::gfp^TCD fluorescence output presented in Figure 2.

https://doi.org/10.1371/journal.pone.0084382.g003

All three constructs demonstrated the classic peak in expression during exponential growth. However, Pfis(−198) and Pfis(−49) showed reduced overall expression levels at all stages of growth in both aerated and non-aerated conditions (Figure 3B), indicating that the full 300-bp upstream region contributes to activation. Furthermore, the minimal RpoD-driven promoter, Pfis(−49), was sufficient for strong induction during exponential growth in S. Typhimurium, as reported in E. coli [6,8]. In non-aerated conditions, sustained fis expression was lower in Pfis(−198) and was even further reduced in Pfis(−49) (Figure 3B), indicating that one or more regulatory elements upstream contribute to sustained expression.

Transcription factor control of S. Typhimurium fis expression

To determine which transcription factors contribute to sustained fis expression and to identify regions of the fis promoter required for transcription factor function, Pfis truncates were tested in several mutant backgrounds. In E. coli, FIS represses its own promoter by binding the FIS-I and FIS-II binding sites that flank the transcription start site [4,6], and these sites are perfectly conserved between the E. coli and S. Typhimurium fis promoters (Figure 1). Up-regulation of the S. Typhimurium fis promoter in the Δfis mutant confirmed that FIS represses its own promoter, and this repression occurs in both aerated and non-aerated conditions (Figure 3C & D). The FIS-I and FIS-II sites are intact in the shortest construct, Pfis(−49), allowing FIS to repress each of the three promoter truncates.

All three lengths of Pfis showed elevated expression in the ΔarcA mutant in non-aerated conditions (Figure 3D). Elevated expression regardless of promoter length supports a model in which ArcA has an indirect influence on fis during stationary phase, consistent with the absence of a predicted ArcA site in the S. Typhimurium fis promoter. fis expression was slightly elevated in aerated conditions (Figure 3C), which was not detected using the chromosomal system — again indicating that the plasmid-borne system enhances or exaggerates fis promoter activity.

The ΔrpoS mutation did not have a detectable effect on the Pfis(−298) and Pfis(−198) truncates, as observed above with the dusB-fis-gfp^TCD chromosomal fusion (Figure 3C & D). However, Pfis(−49) revealed that RpoS can exert a mild repressive effect in the absence of upstream fis promoter DNA, both in aerated and non-aerated conditions.

CRP is a global-acting transcription factor that directly regulates FIS expression in E. coli [4]. We found that deletion of crp caused a slight reduction in fis expression from all three promoter truncates in both aerated and non-aerated conditions (Figure 3C & D). The absence of a DNA site matching the CRP binding site consensus (Figure 1) suggest that CRP plays an indirect regulatory role at the fis promoter in S. Typhimurium.

The S. Typhimurium fis regulatory region has a second, anaerobically-induced promoter

The reduction in fis expression caused by progressive truncation of the fis promoter could be explained by removal of one or more upstream transcription start sites (TSS). Thus, we used recently published RNA-seq data [24] to search for evidence of transcripts originating upstream of the primary TSS, which revealed a putative TSS at position −216 relative to the primary TSS (Figure 1). 5′-RACE confirmed the existence of the second TSS (Figure 4A), which we named Pfis-2 to distinguish it from the gene proximal TSS, called Pfis-1. Pfis-2 was not included in Kröger et al. [24] because it did not pass the conservative threshold used in that study.

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Figure 4. Characterisation of S. Typhimurium Pfis-2.

A) RNA-seq and 5′-RACE identification of the Pfis-2 transcription start site. The number of sequencing reads in RNA-seq analysis of an early stationary phase S. Typhimurium culture is aligned with DNA sequencing results from 5′-RACE analysis of the same RNA sample (RNA-seq data available in [24]). B) Quantitative PCR measurement of transcripts originating from Pfis-1 and Pfis-2 during exponential growth. C) Quantitative PCR measurement of rpoS expression. D) Quantitative PCR measurement of transcripts originating from Pfis-1 and Pfis-2 during anaerobic shock. E) Quantitative PCR measurement of rpoS expression. In B-E, the mean and standard deviation from 3 to 5 biological replicates are plotted.

https://doi.org/10.1371/journal.pone.0084382.g004

To determine the activity of Pfis-2 in laboratory culture, quantitative PCR was used to distinguish between transcripts originating from Pfis-1 and Pfis-2. During exponential growth, less than one percent of dusB-fis transcripts originated at Pfis-2 (Figure 4B). Transcription from Pfis-2 was up-regulated two-fold in non-aerated stationary phase cultures, but this accounted for only 7±1% of total fis transcripts, indicating that Pfis-2 is not responsible for sustained fis expression. Transcription was moderately reduced at both Pfis-1 and Pfis-2 in the ΔarcA mutant (Figure 4B), suggesting that ArcA causes mild activation of fis expression during exponential growth. Deletion of rpoS revealed RpoS to be a weak repressor of Pfis-1 and a stronger repressor of Pfis-2 during exponential growth (Figure 4B).

To further test whether ArcA acts indirectly through RpoS by repressing rpoS transcription, we measured rpoS transcript abundance. No significant difference was detected in rpoS transcript levels between wild type and ΔarcA mutant cells in exponential growth (Figure 4C). RpoS is controlled primarily by post-transcriptional mechanisms; unfortunately attempts to quantify RpoS protein levels in the wild type and ΔarcA mutant in exponential growth were unsuccessful because RpoS protein levels were below the detection limit of western blot assays.

Two-fold up-regulation of Pfis-2 in non-aerated growth conditions led us to test the effects of more severe anaerobic shock, a condition that, like non-aerated growth, may imitate conditions encountered by S. Typhimurium in the intestine and in intracellular environments. Exponentially growing cells were transferred to sealed tubes to induce anaerobic shock, and this caused a significant up-regulation of Pfis-2 concomitant with 18-fold repression of Pfis-1 (Figure 4D). In these conditions, deletion of arcA caused a decrease in Pfis-1 and Pfis-2 activity, consistent with the decrease in exponential phase fis expression seen in Figure 2D. To once again test whether the ArcA effect might arise through RpoS activity, rpoS expression was measured in anaerobic shock. ArcA was found to exert a repressive effect on rpoS as revealed by higher rpoS transcript levels in the ΔarcA mutant (Figure 4E). Although higher levels of RpoS protein in the ΔarcA mutant could explain the repression of Pfis-2 observed in Figure 4C, the ΔrpoS mutant showed wild type levels of Pfis-2 expression during anaerobic shock (Figure 4D). The absence of an explicit RpoS effect in aerated, non-aerated, and anaerobic conditions (Figures 2E, 4C & D), suggests a model in which RpoS represses fis expression only when RpoS protein levels are unusually high, as in a ΔarcA mutant.

DNA supercoiling control of S. Typhimurium fis expression

DNA supercoiling is a key driver of fis expression [9], raising the intriguing question of whether DNA supercoiling and the fis promoter respond to small incremental changes in oxygen availability. Conversely, there may be specific oxygen concentrations at which regulatory control undergoes a transition that facilitates sustained fis expression in non-aerated conditions. To assess the relationship between oxygen availability, DNA supercoiling, and fis promoter activity, aeration was adjusted by growing cells in tubes with increasing culture volumes. DNA supercoiling and fis expression were measured in parallel cultures containing either pUC18 or the Pfis(-298) reporter plasmid, respectively. The small, high copy number plasmid pUC18 (2,686 bp) was ideal for quantifying supercoiling levels, whereas the larger (6,424 bp) and low copy number reporter plasmid pZec-Pfis was less reliable for resolution of topoisomers; nevertheless, we did find that pZec-Pfis had the same topological responses as pUC, as was previously confirmed for pZec [26]. In the present study, wild type cells carrying pUC18 were assayed in parallel with strains having either the plasmid-borne Pfis(-298) reporter or the chromosomal dusB-fis-gfp^TCD reporter.

A continuous increase in DNA supercoiling was observed as aeration decreased due to increased culture volume (Figure 5A). In these same conditions Pfis expression demonstrated a gradual increase with decreased aeration. Increased expression was observed with both the plasmid-borne and chromosomal reporters, but as expected was more pronounced in the plasmid system. Pfis expression was the same in 6 ml and the 10 ml “non-aerated” condition used in the experiments above (compare Figures 3D and 5A); thus cells appear to be severely oxygen limited in volumes above 5 ml in standard culture tubes. These results suggest that changes in DNA supercoiling during non-aerated growth may be involved in sustained fis expression.

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Figure 5. DNA supercoiling control of fis expression.

A) Median and interquartile ranges of DNA supercoiling from four biological replicates, plotted as in Figure 4E. Fluorescence data from Pfis(-298) (green circles) and dusB-fis::gfp^TCD (blue diamonds) for each biological replicate is plotted on the right y-axis; units as in Figure 2. For both the DNA supercoiling and gene expression measurements, wild type cells were grown to stationary phase cells in the indicated volume of culture medium. The dashed lines are nonlinear curves fit to the expression data, with goodness-of-fit R² >0.85 in both cases. The degree of DNA supercoiling (σ) was determined by measuring the migration of topoisomers relative to fully relaxed DNA in chloroquine gels; each topoisomer represents a change of 1 in the linking number. B) DNA supercoiling states in S. Typhimurium wildtype and ΔarcA mutant cells at 22 hours in non-aerated conditions. Medians (black bar) and interquartile ranges of pUC18 topoisomer distributions in stationary phase in well-aerated cultures. For each strain, the average interquartile range from four biological replicates is plotted. C) SIDD profile of the fis promoter region. The energy required for DNA strand separation at a base pair, G(x), is a function of adjacent and distant DNA sequence [30], and G(x) values below 10 indicate positions prone to SIDD. G(x) values for linear DNA were calculated by WebSIDD [27] using 3,500 bp of chromosomal DNA sequence on either side of the dusB start codon (7,000 bp total); only the 550 bp region containing Pfis-1 and Pfis-2 is shown.

https://doi.org/10.1371/journal.pone.0084382.g005

The correlation between DNA supercoiling and sustained fis expression in non-aerated cultures prompted us to test whether increased fis expression in the ∆arcA mutant might also correlate with an increase in DNA supercoiling in this genetic background. DNA supercoiling was not elevated in the ΔarcA mutant (Figure 5B).

DNA supercoiling reduces the amount of energy required for transcription initiation by exerting torsional stress on the DNA double helix, which weakens base pairing and facilitates melting; this is referred to as stress-induced duplex destabilization (SIDD) [30]. Because DNA supercoiling stimulates fis expression we predicted that the fis promoter region would have a SIDD profile indicative of significant stress-induced denaturation, which may be focused near Pfis-1 and Pfis-2. Figure 5C plots the predicted stability of the fis promoter at the supercoiling level observed both in exponential growth and in non-aerated stationary phase (superhelix density around -0.05). This analysis suggests that the S. Typhimurium fis promoter region is particularly prone to destabilization and melting when DNA is highly supercoiled. The region immediately upstream of Pfis-1 (position −10 to −160) is predicted to be extremely destabilized, with a second smaller region of destabilization surrounding Pfis-2 (position −190 to −260). This very strong SIDD profile raises the possibility that DNA supercoiling alone is able to activate transcription. Further, the observed SIDD profile helps explain why removal of the most destabilized region makes the Pfis(−49) truncate deficient in sustained expression.