In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

The RpoS/σS sigma subunit of RNA polymerase is the master regulator of the general stress response in many Gram-negative bacteria. Extensive studies have been conducted on σS-regulated gene expression at the transcriptional level. In contrast, very limited information regarding the impact of σS on global protein production is available. In this study, we used a mass spectrometry-based proteomics approach to explore the wide σS-dependent proteome of the human pathogen Salmonella enterica serovar Typhimurium. Our present goals were twofold: (1) to survey the protein changes associated with the ΔrpoS mutation and (2) to assess the coding capacity of σS-dependent small RNAs. Our proteomics data, and complementary assays, unravelled the large impact of σS on the Salmonella proteome, and validated expression and σS regulation of twenty uncharacterized small proteins of 27 to 96 amino acids. Furthermore, a large number of genes regulated at the protein level only were identified, suggesting that post-transcriptional regulation is an important component of the σS response. Novel aspects of σS in the control of important catabolic pathways such as myo-inositol, L-fucose, propanediol, and ethanolamine were illuminated by this work, providing new insights into the physiological remodelling involved in bacterial adaptation to a non-actively growing state.

In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σS accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σS binding to the Crl protein increases σS activity by favouring its association to E. Taking advantage of evolution of the σS sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σS to the S. Typhimurium σS-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σS and to generate in silico models of the σS-Crl complex constrained by mutational analysis. The σS-Crl models suggest that the identified arginine in σS interacts with an aspartate of Crl that is required for σS binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σS-Crl complex.

The RpoS/σS sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σS-dependent control, that of a repressor. Negative regulation by σS has been proposed to result largely from competition between σS and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σS binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σS protein proficient for EσS complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σS requires its binding to DNA. Although the mechanisms of repression by σS are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by EσS.