Biology subjects

Beeler, S. M.

Publications and source records attributed to Beeler, S. M..

3 recordsLinked to original sources

bioRxiv2020

Deciphering the regulatory genome of Escherichiacoli, one hundred promoters at a time

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 {approx} 65% of the promoters we remain completely ignorant of their regulation. Until we have cracked this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method (Reg-Seq) linking a massively-parallel reporter assay and mass spectrometry to produce a base pair resolution dissection of more than 100 promoters in E. coli in 12 different growth conditions. First, we show that our method recapitulates regulatory information from known sequences. Then, we examine the regulatory architectures for more than 80 promoters in the E. coli genome which previously had no known regulation. In many cases, we also identify which transcription factors mediate their regulation. The method introduced here clears a path for fully characterizing the regulatory genome of model organisms, with the potential of moving on to an array of other microbes of ecological and medical relevance.

biophysics↗

bioRxiv2019

The transcriptional response of genes to RpoS concentration in Escherichia coli is not determined by core promoter sequences

The alternative sigma factor RpoS is an important regulatory protein in Escherichia coli, responsible for mediating the general stress response. RpoS levels vary continuously in response to different stresses. Previous work has shown that genes vary in their responsiveness to increasing RpoS concentrations, with some genes being \"sensitive,\" requiring only a low level of RpoS to be relatively highly expressed, while other genes are \"insensitive,\" only being highly expressed in the presence of high levels of RpoS. In other systems, this type of variation is caused by interactions between the regulatory protein and the DNA it binds. To see if this is the case for RpoS, we measured twelve RpoS binding site mutants for their effects on maximal expression and responsiveness to increasing RpoS concentration. While maximal expression varied over an order of magnitude across these twelve constructs, the responsiveness to increasing RpoS concentration was largely unaffected, suggesting that the RpoS binding site alone is not responsible for a genes sensitivity or insensitivity to RpoS. In addition, we swapped the RpoS binding region between sensitive and insensitive promoters and found no change in the behavior of the promoter. Taken together, these results argue that differences in sensitivity of the RpoS-dependent promoters are not due to interactions between RpoS and the various DNA sites it binds.

microbiology↗

bioRxiv2016

The genome-wide transcriptional response to varying RpoS levels in Escherichia coli K-12

The alternative sigma factor RpoS is a central regulator of a 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 show that 23% of genes in the E. coli genome are regulated by RpoS level, and we identify many RpoS-transcribed genes and promoters. We observe 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 a RpoS-regulated gene in 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.\n\nImportanceThe 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 to coordinate specific cellular processes in response to stresses.

microbiology↗