Novel DNA Binding and Regulatory Activities for σ54 (RpoN) in Salmonella enterica Serovar Typhimurium 14028s

Abstract

The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. Salmonella enterica serovar Typhimurium utilizes the housekeeping σ⁷⁰ and five alternative sigma factors, including σ⁵⁴ The σ⁵⁴-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In S. Typhimurium, σ⁵⁴-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ⁵⁴-RNAP DNA binding sites and the transcriptome of the σ⁵⁴ regulon of S. Typhimurium. The position and context of many of the identified σ⁵⁴ RNAP DNA binding sites suggest regulatory roles for σ⁵⁴-RNAP that connect the σ⁵⁴ regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions.IMPORTANCE The alternative sigma factor σ⁵⁴ (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ⁵⁴ regulon of the versatile pathogen S. Typhimurium has expanded our understanding of the scope of the σ⁵⁴ regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.

Keywords: RpoN; Salmonella enterica serovar Typhimurium; bEBP; promoter; regulon; σ54.

FIG 1

Graphical representations of multiple sequence alignments for S. Typhimurium 14028s Eσ⁵⁴ binding sites. The relative frequency of bases at a given position is illustrated by WebLogo (100, 101) for multiple sequence alignments of (A) the 18-bp core sequences (−9 to −26 from the transcriptional start) of 33 σ⁵⁴-dependent promoters in Salmonella (, ; this study), (B) the 52 Eσ⁵⁴ binding sites used for generating the PSSM for Eσ⁵⁴ binding site prediction (see Materials and Methods); (C) the 186 predicted Eσ⁵⁴ binding sites from the ChIP-chip analysis; and (D) the 68 predicted Eσ⁵⁴ binding sites from the ChIP-chip analysis that have PSSM scores of >14.

FIG 2

PSSM score distribution for 18-mer potential Eσ⁵⁴ binding sites in S. Typhimurium and randomized genome sequences. The ordinate shows the number of 18-mers found in the sequence with PSSM scores greater than or equal to the cutoff shown on the abscissa. Note that the plot displays only the right tail of the whole PSSM score distribution (scores of ≥0) and the 55,840 18-mers that yield scores of ≥0 represent only 0.57% of all 9,740,530 18-mers in the genome (in both DNA strands). The blue line refers to the S. Typhimurium genome. The thick black line signifies the median value among 1,000 randomized genome sequences, and the thin black lines correspond to the 1st, 5th, 25th, 75th, 95th, and 99th percentiles.

FIG 3

Illustrations of contextual positions of Eσ⁵⁴ DNA binding sites in the S. Typhimurium genome and examples of potential regulatory roles with corresponding plots of microarray data. (A) The 186 Eσ⁵⁴ binding sites that were identified in the ChIP-chip analysis are grouped into classes A to F by position and orientation (blue arrows) relative to annotated ORFs (gray arrows), as described in the Results section. Sequences of these binding sites are given in Table S2. (B) Six examples of potential regulatory roles for the Eσ⁵⁴ binding sites are illustrated with the relative transcript levels (ratio of WT DctD250 to ΔrpoN DctD250 mutant), as determined by microarray (red numbers and arrows for σ⁵⁴-dependent gene transcripts, green numbers and arrows for σ^70/38-dependent transcripts) and qRT-PCR (σ⁵⁴-dependent gene transcripts in red brackets, σ⁷⁰-dependent gene transcripts in green brackets). The dashed green arrows indicate the σ⁷⁰-dependent gene transcripts that are significantly downregulated, but less than 2-fold, in the presence of RpoN. The fold enrichment in WT DctD250 versus the ΔrpoN DctD250 mutant in ChIP-chip analysis for each Eσ⁵⁴ binding site is shown in blue, and an asterisk indicates that the binding site was confirmed by EMSA (Table 3). Primary and secondary promoter designations are from Kröger et al. (2). Adjacent to each example is the WebArrayDB plot of expression microarray data for the genes whose transcription is positively or negatively regulated by σ⁵⁴. Each dot, which is the log₂-transformed ratio (WT DctD250 to ΔrpoN DctD250 mutant) for each probe (for all 3 biological replicates), is plotted on the x axis by genome position. (Nucleotide positions are not shown.) The dot colors indicate probe orientation and significance of P values; red is positive strand with a significant P value, light pink is a positive strand without a significant P value, dark blue is a negative strand with a significant P value, and light blue is a negative strand without a significant P value. The upward and downward carets designate the start and end, respectively, of a gene. (The name or 14028s locus number is given.)

FIG 4

PSSM score distribution for intragenic 18-mer potential Eσ⁵⁴ binding sites on the coding and template strands of S. Typhimurium and randomized gene sequences. The ordinate shows the number of 18-mers with PSSM scores greater than or equal to the cutoff shown on the abscissa for the coding strands (solid lines) and template strands (dashed lines) of S. Typhimurium protein-coding sequences (blue lines) or randomized protein-coding sequences (median values, black lines).

FIG 5

In vitro assays of Eσ⁵⁴ and σ⁵⁴ binding to DNA sequences identified in ChIP-chip analysis of Eσ⁵⁴ genomic binding sites. (A) Representative EMSAs for binding reaction mixtures containing 16 nM ³²P-labeled 50-bp heteroduplex oligonucleotide probes (*P) for the positive control (nifH049 promoter) and the intragenic Eσ⁵⁴ binding sites in proP and STM14_0816 with 0, 10, 50, 100, or 200 nM Eσ⁵⁴, 100 nM core RNAP, or 100 nM, 500 nM, 1 μM, or 2 μM σ⁵⁴ protein, unbound probe, and the protein-DNA complexes, Eσ⁵⁴-*P, σ⁵⁴-*P, or core-*P (marked by arrows) separated by native PAGE (6.5% acrylamide). Images are from Typhoon scans of gel-exposed phosphorimager screens. (B) Examples of ChIPeak output from analysis of Eσ⁵⁴ ChIP-chip data show the peaks for enriched probes within proP and STM14_0816 and in the intergenic regulatory region between hypO and yghW.

FIG 6

Consensus sequence for σ⁵⁴ binding in the absence of RNAP core. Alignment of the sequence from −40 to +10 (relative to the +1 transcription start site) of the σ⁵⁴-dependent promoters K. pneumoniae (K. p.) nifH049 and S. meliloti (S. m.) nifH, which were previously shown to bind σ⁵⁴ in the absence of RNAP (40, 70), with the newly identified S. Typhimurium (S.T.) proP and STM14_0816 σ⁵⁴ binding sites (Table 3). The −24 GG and −12 GC promoter elements are in boldface. The extent of the DNase I footprint for σ⁵⁴ in the closed complex with nifH049 and S. meliloti nifH (40, 70) is indicated by the black bar underneath the sequence. The consensus sequence was generated for the four σ⁵⁴ binding sites using the single-letter codes for nucleotides as defined by NCBI: M, A/C; R, A/G; W, A/T; S, C/G; Y, C/T; K, G/T; V, not T; H, not G; D, not C; B, not A; and N, any nucleotide. The consensus σ⁵⁴ binding sequence is aligned with the inter-hypO-yghW sequence that does not bind σ⁵⁴ but has the −14 to −17 T-tract previously proposed to be associated with σ⁵⁴ binding (40) and the same bases at the DNA distortion in the probes used for EMSA as nifH049 and proP (Table 3). Nucleic acid residues shared between the consensus sequence and nonbinding sequence are struck through.