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

In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parentspecific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species.

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.

The epigenetic mark of DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3′ direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.

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