Abstract
Escherichia coli cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard E. coli K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to E. coli K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.
Highlights
During stress conditions, cells of Escherichia coli (E. coli) impose dramatic changes in their transcriptional profile and proteome to combat stressors
We added spike-in cells to the experimental samples prior to total RNA extraction to ensure that variations in RNA recovery, complementary DNA (cDNA) synthesis, and sequencing depth between the samples would be reflected in the numbers of spike-in reads
We were able to, very precisely, quantify the relative changes in the transcriptome during the experiment, while we lost the information about absolute amounts of transcripts mapped, which is only obtainable by addition of in vitro transcribed spike-in RNA after sample preparation (Gorochowski et al, 2019)
Summary
Cells of Escherichia coli (E. coli) impose dramatic changes in their transcriptional profile and proteome to combat stressors. The cells ensure that genes important to overcome the stress are turned on and other redundant and energy-demanding gene products, such as genes of the protein synthesis machinery [i.e., those encoding the ribosomes, transfer RNAs (tRNAs) and factors required for translation] are downregulated. In E. coli, the synthesis of ppGpp is mediated by two related proteins, RelA and SpoT; each requiring different signals for activation. The RelA protein is associated with uncharged tRNA and the synthesis of ppGpp is triggered when the translating ribosome binds a RelA-tRNA complex at the starving A-site codon (Haseltine and Block, 1973; Winther et al, 2018). The SpoT protein is bi-functional; besides synthesizing ppGpp, SpoT can hydrolyse ppGpp to guanosine diphosphate and pyrophosphate (Murray and Bremer, 1996), allowing a way out of stringency when conditions allow
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