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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. Author summary: RpoD-family sigma factors in Salmonella enterica serovar Typhimurium orchestrate transcription initiation for diverse metabolic genes in response to environmental stimuli. Heat stress, a fundamental challenge for bacteria in both natural and industrial settings. Typically triggers the up-regulation of heat shock proteins that assist in protein folding and degradation of damaged proteins. However, the full scope of transcriptional changes induced by heat shock extends beyond this familiar response. In this study, we employed systems biology approaches to elucidate detailed alterations in transcriptional regulatory networks (TRNs) mediated by RpoD-family sigma factors in response to heat shock. Machine learning-based transcriptomic analysis broke down the components of heat stress, revealing its involvement in both anaerobic and oxidative stress responses. By integrating genome-scale data with this nuanced understanding of heat shock, we reconstructed the TRNs and uncovered several key findings: (i) A competition mechanism exists between RpoS and RpoD at overlapping binding sites upstream of promoter region in response to heat shock; (ii) Heat-induced anaerobic stress down-regulated the majority of electron transporter genes, leading to significant modifications in the TRN governing carbon metabolism; (iii) Under heat shock conditions, alterations in iron availability induced down-regulation of extracellular iron uptake and enterobactin systems, while up-regulating intracellular iron-utilizing genes. This regulatory response may reduce intracellular iron concentrations, potentially attenuating Fenton reaction-mediated oxidative stress. These comprehensive results illuminate how RpoD-family sigma factors in S. Typhimurium regulate diverse cellular processes to adapt to heat shock, encompassing responses to anaerobic and oxidative stress. Our findings provide a more holistic understanding of bacterial heat shock response, extending beyond the classical heat shock protein paradigm. [ABSTRACT FROM AUTHOR]