The RpoS/σS sigma subunit of RNA polymerase (RNAP) controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses. σS also contributes to biofilm formation and virulence of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). In this study, we used directional RNA-sequencing and complementary assays to explore the σS-dependent transcriptome of S. Typhimurium during late stationary phase in rich medium. This study confirms the large regulatory scope of σS and provides insights into the physiological functions of σS in Salmonella. Extensive regulation by σS of genes involved in metabolism and membrane composition, and down-regulation of the respiratory chain functions, were important features of the σS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions. As an example, we show that arginine catabolism confers a competitive fitness advantage in stationary phase. This study also provides a firm basis for future studies to address molecular mechanisms of indirect regulation of gene expression by σS. Importantly, the σS-controlled downstream network includes small RNAs that might endow σS with post-transcriptional regulatory functions. Of these, four (RyhB-1/RyhB-2, SdsR, SraL) were known to be controlled by σS and deletion of the sdsR locus had a competitive fitness cost in stationary phase. The σS-dependent control of seven additional sRNAs was confirmed in Northern experiments. These findings will inspire future studies to investigate molecular mechanisms and the physiological impact of post-transcriptional regulation by σS.
Proteins that bind factors typically attenuate the function of the factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor S (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of S -dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a ⌬crl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-S interaction and that residues 1 to 71 in S are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.In bacteria, transcription depends on a multisubunit RNA polymerase (RNAP) consisting of a catalytically active core enzyme (E) with a subunit structure ␣ 2 Ј that associates with any one of several factors to form different holoenzyme (E) species. The subunit is required for specific promoter binding, and different factors direct RNAP to different classes of promoters, thereby modulating the gene expression patterns (17). The RNA polymerase holoenzyme containing the 70 subunit is responsible for the transcription of most genes during exponential growth (17). When cells enter stationary phase or are under specific stress conditions (high osmolarity, low pH, or high and low temperatures) during exponential growth, S , which is encoded by the rpoS gene, becomes more abundant, associates with the core enzyme, and directs the transcription of genes essential for the general stress response and for stationary phase survival (17,20,25).Sigma factors compete for binding to a limited amount of the core polymerase (16,17,20,34).70 is abundant throughout the growth cycle and has the highest affinity of all sigma factors for E in vitro (20). In contrast, levels of S reach only about one-third of the 70 levels upon entry into stationary phase, and S exhibits the lowest affinity for E of all sigma factors in vitro (20). The cell uses at least two strategies to ensure the switch between 70 -and S -associated RNA polymerases and to allow gene expression to be reprogrammed upon entry into stationary phase. Several factors (Rsd, 6S RNA, ppGpp, and D...
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.
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