The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS. IMPORTANCEThe sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli. Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses. KEYWORDS RpoS, promoters, S , stress response, transcriptional regulation, transcriptome G enome-wide measurements of RNA levels have revolutionized our understanding of how cells organize their patterns of transcription. These studies have given us snapshots of how patterns of gene expression change in response to changes in the external environment. They have also allowed us to define the regulons controlled by specific transcription factors (TFs). A major weakness of the vast majority of these

The bacterial predator Bdellovibrio bacteriovorus is evolved to attack and kill other bacteria, including the human intestinal pathogen Vibrio cholerae. Although B. bacteriovorus exhibit a broad prey range, little is known about the genetic determinants of prey resistance and sensitivity. Here we perform a genetic screen on V. cholerae and identify five pathways contributing to predation susceptibility. We find that the essential virulence regulators ToxR/S increase susceptibility to predation, as mutants of these genes are more resistant to predation. We observe by flow cytometry that lipopolysaccharide is a critical defense, as mutants lacking O-antigen are rapidly attacked by predatory B. bacteriovorus. Using polymer solutions to alter media viscosity, we find that when B. bacteriovorus attacks motile V. cholerae, increased drag forces slow its ability to prey. These results provide insights into key prey resistance mechanisms, and may be useful in the application of B. bacteriovorus in treating infections.

DNA–protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA–protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA–protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

28The alternative sigma factor RpoS is a central regulator of a many stress responses in 29Escherichia coli. The level of functional RpoS differs depending on the stress. The effect 30 of these differing concentrations of RpoS on global transcriptional responses remains 31 unclear. We investigated the effect of RpoS concentration on the transcriptome during 32 stationary phase in rich media. We show that 23% of genes in the E. coli genome are 33 regulated by RpoS level, and we identify many RpoS-transcribed genes and promoters. 34We observe three distinct classes of response to RpoS by genes in the regulon: genes 35 whose expression changes linearly with increasing RpoS level, genes whose 36 expression changes dramatically with the production of only a little RpoS ("sensitive" 37 genes), and genes whose expression changes very little with the production of a little 38RpoS ("insensitive"). We show that sequences outside the core promoter region 39 determine whether a RpoS-regulated gene in sensitive or insensitive. Moreover, we 40 show that sensitive and insensitive genes are enriched for specific functional classes, 41and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells 42 enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to 43 coordinate specific cellular processes with growth phase, and may also contribute to the 44 diversity of stress responses directed by RpoS. 45 46Importance 47 The sigma factor RpoS is a global regulator that controls the response to many stresses 48 in Escherichia coli. Different stresses result in different levels of RpoS production, but 49 the consequences of this variation are unknown. We describe how changing the level of 50 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a