Unveiling the novel regulatory roles of RpoD-family sigma factors in Salmonella Typhimurium heat shock response through systems biology approaches

Abstract

Three RpoD-family sigma factors, RpoD, RpoS, and RpoH, play critical roles in transcriptional regulation in Salmonella enterica serovar Typhimurium under heat shock conditions. However, the genome-wide regulatory mechanisms of these sigma factors in response to heat stress have remained elusive. In this study, we comprehensively identified 2,319, 2,226, and 213 genome-wide binding sites for RpoD, RpoS, and RpoH, respectively, under sublethal heat shock conditions (42°C). Machine learning-based transcriptome analysis was employed to infer the relative activity of iModulons, providing valuable insights into the transcriptional impact of heat shock. Integrative data analysis enabled the reconstruction of the transcriptional regulatory network of sigma factors, revealing how they modulate gene expression to adapt to heat stress, including responses to anaerobic and oxidative stresses. Notably, we observed a significant expansion of the RpoS sigmulon from 97 to 301 genes in response to heat shock, underscoring the crucial role of RpoS in regulating various metabolic processes. Moreover, we uncovered a competition mechanism between RpoD and RpoS within RpoS sigmulons, where RpoS significantly increases its binding within promoter regions shared with RpoD under heat shock conditions. These findings illuminate how three RpoD-family sigma factors coordinate multiple cellular processes to orchestrate the overall response of S. Typhimurium to heat stress.

Fig 1. Overview of the reconstruction of Salmonella transcriptional regulatory network (TRN).

(A) Schematic representation of TRN reconstruction for the heat shock response in S. Typhimurium 14028s. RpoS binding sites and transcript expression profiles of the ΔrpoS mutant are used to define the RpoS sigmulons, representing the set of genes directly regulated by RpoS. Binding sites of RpoD and RpoH, along with their expression changes upon heat shock, are used to establish both sigmulons. Additionally, a strain-specific RNA-seq compendium for S. Typhimurium 14028s is utilized to infer the activity levels of each iModulon, guiding predictions of changes in TRNs in response to heat shock. (B) COG analysis on up- and down-regulated differentially expressed genes (DEGs) in response to heat shock. Up-regulated DEGs have functions enriched in “Intracellular trafficking, secretion vesicular transport”, “Energy production conversion”, and “Carbohydrate transport metabolism” categories (Hypergeometric test p-value < 0.05). Down-regulated DEGs have functions enriched in “Cell wall/membrane/envelope biogenesis, “Cell motility”, “Translation, ribosomal structure biogenesis”, “Amino acid transport metabolism”, and “Nucleotide transport metabolism” categories (Hypergeometric test p-value < 0.05). (C) Bar plots depict the relative activity of RpoS and RpoH iModulons under heat shock conditions (mid-exponential phase at 42°C). The asterisk indicates the control conditions (mid-exponential phase at 37°C).

Fig 2. Changes in the heat shock TRNs regulated by RpoD-family sigma factors.

(A) Venn diagrams show overlap of binding sites for each sigma factor before and after heat shock treatment. (B) Motif analysis of RpoS, RpoD and RpoH binding sites identified by ChIP-mini under both conditions. (C) Comparison of sigma factor binding results and differentially expressed genes (DEGs) to define sigmulon genes. Core DEGs represent heat shock DEGs found in the target genes with sigma factor binding under both control and heat shock conditions. Unique DEGs are those observed in target genes with sigma factor binding occurring exclusively under either control or heat shock conditions. Genes with expression changes with an absolute value of log₂ fold change ≥ 1.0 and a false discovery rate < 0.05 are defined as differentially expressed.

Fig 3

Genome-wide identification of RpoS sigmulons in response to heat shock (A) The scatter plot illustrates the fold changes in intensity of overlapping binding sites between RpoD and RpoS in response to heat shock. The grey dotted line indicates trend line for all overlapping binding sites, while the red dotted line denotes the trend line for overlapping binding sites with significant changes in binding intensity (absolute value of log₂ fold change ≥ 1.0 and false discovery rate < 0.05). Red and blue circles represent up-regulated and down-regulated DEGs in response to heat shock, respectively. (B) Comparison of RpoS sigmulons between control and heat shock conditions. In response to heat shock, the number of RpoS sigmulon genes increased approximately threefold. The majority of sigmulon genes found under control conditions overlap with those under heat shock conditions. Under heat shock, RpoS regulates a total of 301 genes, including carbon metabolism and electron transport chain (24 genes), iron metabolism (9 genes), stress-related proteins (16 genes), ppGpp metabolism (2 genes), superoxide radicals degradation (2 genes), virulence (3 genes), transcription factors and sigma factors (21 genes), two-component systems (2 genes), flagellar and fimbriae synthesis (4 genes), amino acid/amine degradation (5 genes), biofilm synthesis (3 genes), transporter subunits (39 genes), metabolic processes (72 genes), and other functions (99 genes). (C) Overlap of target genes for RpoD and RpoS within RpoS sigmulon genes under heat shock conditions. (D) Changes in binding intensity within RpoS sigmulons before and after heat shock. The binding intensity of RpoS at promoter regions was similar between control and heat shock conditions when bound only by RpoS. In contrast, the binding intensity of sites bound by RpoD and RpoS increased in response to heat shock.

Fig 4

Transcriptional regulation of heat shock-related protein genes by RpoH during heat shock conditions (A) Comparison of RpoH target DEGs between control and heat shock conditions. (B) Overlap of RpoH target genes with those of other sigma factors, illustrating the intersection of RpoH-bound genes with RpoD and RpoS sigmulons under both control and heat shock conditions. (C) The mRNA expression levels of heat shock-related protein genes were assessed before and heat shock treatment. Genes significantly upregulated in response to heat shock are indicated with three asterisks (log₂ fold change ≥ 1.0 and false positive rate <0.001). (D) Heat map showing sigma factor binding and expression changes for heat shock-related protein genes in response to heat shock. The upper three stacks denote the binding of each sigma factor (RpoD, RpoS, and RpoH), and the bottom stack indicates the relative expression changes of these genes between heat shock (42°C) and control conditions (37°C). (E) Box plot illustrates the intensity of RpoH binding observed upstream of heat shock-related protein genes under control and heat shock conditions, showing increased binding intensity in response to heat shock.

Fig 5

Transcriptional regulation of electron transport chain genes in response to heat shock, mirroring anaerobic stress (A) Bar plots depicting the relative activity of the Fnr-1 iModulon under heat shock conditions (mid-exponential phase at 42°C). Red bars denote the activity of Fnr-1 in response to heat shock, inferred using the S. Typhimurium 14028s RNA-seq compendium. Grey bars illustrate the activity of Fnr-1 in response to anaerobic shock and anaerobic growth in the S. Typhimurium core RNA-seq compendium. The asterisk indicates the control conditions (mid-exponential phase at 37°C). (B) Heat map of sigma factor binding and expression change for electron transport chain genes in response to heat shock. Expression levels of cytochrome bo₃, cytochrome bd, ATP synthase, and NADH dehydrogenase I genes are down-regulated by heat shock, while cytochrome bd-II genes (cyxAB) are significantly up-regulated by RpoS under heat shock conditions. The upper three stacks denote the binding of each sigma factor, and the bottom stack indicates relative gene expression changes between heat shock (42°C) and control conditions (37°C). In addition, genes up-regulated by RpoS are shown in red, and those down-regulated by RpoS are represented in blue. (C) RpoD binding events upstream of TUs for cytochrome bo₃, cytochrome bd. The binding intensity of RpoD upstream of cyoABCDE and cydAB decreases in response to heat shock. (D) RpoS binding events upstream of ssaA-cyxAB transcription units (TUs). The binding intensity of RpoS upstream of cyxAB significantly increases in response to heat shock. (E) RpoD binding events upstream of TUs for NADH:quinone oxidoreductase and ATP synthase. Among the three RpoD binding sites upstream of cyoABCDE, only the binding intensity at the site corresponding to the transcription start site (TSS) significantly decreases. Additionally, a significant reduction in RpoD binding is also observed for atpIBEFHAGDC. (E) Differential transcriptional regulation of electron transport chain genes by sigma factors in response to heat shock. This schematic summarizes how heat shock impacts sigma factor binding and gene expression, highlighting the regulatory roles of RpoS and RpoD in modulating the expression of electron transport chain components.

Fig 6

Heat shock induces fluctuations in carbon metabolism in Salmonella (A) Bar plot depicting the relative activity of iModulons related to carbon metabolism including Mlc, MalT, and Cra, under heat shock condition (mid-exponential phase at 42°C). Asterisk indicates the control condition (mid-exponential phase at 37°C). (B) RpoD binding events upstream of the phosphotransferase system (PTS) genes under four conditions. The binding intensity of RpoD upstream of ptsG and ptsHI significantly decreases in response to heat shock. (C) Heat map showing sigma factor binding and expression change for carbon metabolism genes in response to heat shock. RpoS directly up-regulates five isozymes involved in carbon metabolism including glycolysis, the pentose phosphate pathway, and the TCA cycle under heat shock conditions. Additionally, the expression of acetate and lactate metabolism genes is significantly up-regulated. The left two stacks denote the binding of RpoD and RpoS, and the rightmost stack indicates the relative expression of genes between heat shock (42°C) and control conditions (37°C). Genes up-regulated by RpoS are shown in red, and those down-regulated by RpoS are represented in blue. (D) Diagram showing the overall differentially expressed genes involved in carbon metabolism in response to heat shock. Metabolic pathways regulated by RpoS sigmulon genes are highlighted with a bold light blue line.

Fig 7

Regulation of iron acquisition/storage and superoxide radical degradation pathways to mitigate Fenton reaction caused by heat shock (A) Bar plot depicting the relative activity of the Fur iModulon under control conditions (mid-exponential phase at 37°C). The asterisk indicates the control conditions (mid-exponential phase at 37°C). (B) Representation of the iron acquisition (enterobactin biosynthesis and iron/enterobactin transport), iron storage, and FeS assembly pathways. Expression levels of iron acquisition genes are down-regulated, whereas intracellular iron storage and FeS assembly genes are up-regulated in response to heat shock, possibly leading to a reduction in intracellular iron concentration. The upper two stacks denote the binding of RpoD and RpoS, and the bottom stack indicates the relative gene expression changes between heat shock (42°C) and control conditions (37°C). Genes up-regulated by RpoS are shown in red, and those down-regulated by RpoS are represented in blue. (C) Illustration of the superoxide radical degradation pathway and corresponding mRNA expression levels in wild-type and ΔrpoS strains. (D) Schematic diagram illustrating the transcriptional regulation mechanisms that mitigate the Fenton reaction under heat shock conditions. Heat shock can accelerate the Fenton reaction, producing damaging hydroxyl radicals from hydrogen peroxide. This is mitigated by the down-regulation of iron acquisition and the up-regulation of iron storage and superoxide radical degradation, regulated by RpoS and RpoD. 2,3-DHBA: 2,3-dihydroxybenzoic acid, IM: inner membrane, OM: outer membrane.