Figure 1.
ChIP-seq and ChIP-chip data used in this study.
The tracks are (from top): FNR ChIP-seq −O2 (blue) with peaks upstream of a subset of genes labeled, FNR ChIP-chip in Δhns/ΔstpA −O2 (black), σ70 subunit of RNAP ChIP-seq −O2 (green), σ70 subunit of RNAP ChIP-seq +O2 (red), H-NS CHIP-chip +O2 (light purple), H-NS ChIP-chip −O2 (orange), IHF ChIP-chip −O2 (purple), Fis ChIP-seq +O2 [30] (aqua), β subunit of RNAP −O2 (yellow), β subunit of RNAP +O2 (dark purple), and genomic coordinates. Locations of FNR binding sites are also shown: predicted FNR binding sites (red lines), FNR ChIP-seq peaks (black lines), FNR peaks upstream of operons showing a FNR-dependent change in expression (blue lines), FNR peaks co-activated by NarL/NarP (green lines), FNR peaks co-activated by CRP (purple lines), and FNR peaks repressed by Fur (yellow lines).
Figure 2.
Precision-recall curve used to determine the prediction threshold of FNR binding sites and updated FNR PWM.
The precision-recall curve used to determine the optimal threshold for predicting high quality FNR binding sites throughout the genome. The precision and recall values were determined for many ln(p-value) thresholds using the PatSer algorithm and the optimal value is identified by the arrow. The inset shows the FNR position weight matrix (PWM) constructed from the FNR ChIP-seq peak sequences. The height (y-axis) of the letters represents the degree of conservation at that position within the aligned sequence set (in bits), with perfect conservation being 2 bits. The x-axis shows the position of each base (1–14) starting at the 5′ end of the motif.
Figure 3.
ChIP peak height correlated with PWM score and over a range of FNR levels.
A)Correlation between FNR ChIP-seq peak height (read count at the summit of the peak) and the degree of agreement to the FNR PWM at each peak (as scored by PatSer [36], with higher values indicating a better match to the FNR PWM). The line is the best-fit between peak height and PWM score. B) Comparison of the average ChIP-chip peak height for FNR in WT cultures (open symbols) (∼2.5 µM FNR) and PK8263 (Ptac::fnr) cultures (closed symbols) at three [IPTG] concentrations: 4 µM IPTG (∼450 nM FNR), 8 µM IPTG (∼700 nM FNR), 16 µM IPTG (∼1.9 µM FNR). [FNR] determined by quantitative Western blot. Shown are four representative examples from the 39 regions examined (Figure S1). A t-test shows a statistically significant difference in peak average at all genes between 4 µM IPTG and 8 µM IPTG.
Figure 4.
Identification of FNR occupancy in a Δhns/ΔstpA strain compared to WT.
A) Venn diagram showing the overlap of FNR peaks identified only in the WT strain (purple), in both the WT and the Δhns/ΔstpA strains (blue) or only in the Δhns/ΔstpA strain (green). B) Example of a high-quality predicted FNR binding site (blue line) within fimE that showed no FNR binding in the WT strain (blue trace), but did show enrichment of H-NS in the WT strain (purple trace). A FNR ChIP-chip peak was identified in the Δhns/ΔstpA strain (green trace) at the location of the predicted FNR binding site. C) The 193 FNR peaks found only in the Δhns/ΔstpA strain with a statistical increase in FNR occupancy in the Δhns/ΔstpA strain compared to the WT strain (p-value<0.05). Correlation of ChIP-chip peak average (log2(IP/INPUT) average) and the corresponding FNR PWM score (determined by PatSer [36]). D) Correlation of ChIP-chip peak averages (log2(IP/INPUT) average) for FNR ChIP-chip peaks found in both WT and Δhns/ΔstpA strains. Shown are peaks with no statistical difference in occupancy (red points) and those peaks that showed a statistical increase in FNR occupancy (blue points) in the Δhns/ΔstpA strain compared to the WT strain (p-value<0.05).
Figure 5.
The FNR transcriptional network and categories of FNR regulation.
A) Graphical representation of the FNR transcriptional network. FNR is shown in the blue octagon, while other TFs (CRP, Fur, NarL) are shown as purple diamonds. Circles represent operons with an upstream FNR ChIP-seq peak, while squares represent operons indirectly regulated by FNR. Dark blue circles are operons directly dependent on FNR for expression, with the lighter blue circles representing FnrS other TFs (CaiF, BssR, PdhR, GadE) that potentially control the indirect regulon, shown by yellow squares. Red circles are operons known or predicted to be co-regulated by FNR and other TFs, while green circles have other potential regulatory mechanisms with FNR. B) Each box represents different categories of FNR regulation identified in this study. Categories 1 and 2 (upper left and middle boxes) show direct activation and repression of operons by FNR (blue ovals). Category 3 (upper right box) show co-activation by other TFs (red star; e.g. CRP, NarL, NarP) and Category 4 (lower left box) shows TF repression that prevents FNR regulation (green rectangle; e.g. Fur). Category 5 (lower middle box) represents operons with other possible regulatory mechanisms with FNR and Category 6 (lower right box) shows the subset of the indirect regulon affected by other TFs and, for example, by the small, regulatory RNA FnrS (red line).
Table 1.
Operons with an upstream FNR ChIP-seq peak and a FNR-dependent change in expression under GMM.
Table 2.
Operons associated with a FNR ChIP-seq peak and lacking a FNR-dependent change in expression in GMM but are activated by FNR in the presence of NO3−, NO2−, NarL or NarP (Category 3) according to Constantinidou et al. [19].
Table 3.
Operons associated with a FNR ChIP-seq peak and lacking a FNR-dependent change in expression in GMM but are potentially co-activated by CRP and FNR (Category 3).
Table 4.
Operons associated with a FNR ChIP-seq peak and lacking a FNR-dependent change in expression in GMM but are repressed by Fur (Category 4).
Table 5.
Operons lacking a FNR ChIP-seq peak but with a FNR-dependent change in expression in GMM that are known to be regulated through the action of the small regulatory RNA FnrS (Category 6).
Figure 6.
Glycolysis and mixed acid fermentation pathway overlaid with FNR and O2 regulation.
Pathway map showing the glycolysis and mixed acid fermentation pathway overlaid with FNR ChIP-seq peak occupancy and expression changes [124]. Reactions are represented by arrows connecting metabolites and each operon is represented by a box with three ovals. The first oval of each box indicates the presence (blue) or absence (white) of a FNR ChIP-seq peak upstream of that operon. The color of the second oval indicates the impact of FNR on the expression of the operon (red is FNR repression, while green is FNR activation). The color of the third oval indicates the expression under WT aerobic and anaerobic growth conditions (red is WT aerobic expression, while green is WT anaerobic expression). The blue stars indicate newly identified direct targets of FNR regulation within this pathway.
Figure 7.
Representative examples of FNR directly activated and repressed promoters.
Representative examples of FNR directly activated (dmsA, panel A) and repressed (ndh, panel B) promoters confirmed in our study. Shown are the ChIP-seq data traces of anaerobic FNR (blue), anaerobic σ70 (green) and aerobic σ70 (red). ChIP-seq peak heights are represented on the y-axis (log2 kernel density [115]). The chromosomal location is shown on the x-axis with genes represented by arrows pointing in the direction of transcription. Known promoters are represented as red arrows and previously identified FNR binding sites are shown by blue brackets. The inset shows the linear expression levels for each gene from the RNA-seq experiments comparing FNR+ and Δfnr -O2 expression. Linear tag density [125] for each gene is shown on the y-axis.