Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing

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., nitrogendeprivation, 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.

Possible Roles for Basal Levels of (p)ppGpp: Growth Efficiency Vs. Surviving Stress

“…These results suggested that under a starvation stress situation, cells will first try to relocate resources to restore the normal growth, but as the amounts of (p)ppGpp keep rising, they will go into survival mode. Lrp-dependent regulon gene expression was not seen in the RNA sequencing studies during abrupt starvation of isoleucine by adding valine as noted by Gummesson et al (2020) . Moreover, abrupt starvation provokes unique gene regulation.…”

“…Moreover, abrupt starvation provokes unique gene regulation. such as crp , that increases at the 5 min peak of high (p)ppGpp levels during abrupt starvation, which is followed by lowering of gene expression to pre-induction levels or even lower ( Gummesson et al, 2020 ).…”

“…In MOPS with all the amino acids and IPTG induced RelA, a burst of (p)ppGpp occurs without starvation. When Gummesson et al (2020) compared their results with those of Sanchez-Vazquez et al (2019) ; they found big differences on the expression levels of genes involved in amino acid biosynthesis that they attributed to the media conditions. This suggests that, although in both experiments a burst of (p)ppGpp is being produced, the media conditions will affect the outcome.…”

Distinct survival, growth lag, and ribosomal RNA degradation kinetics during long-term starvation for carbon or phosphate

“…Since the efficiency of RNA extraction can vary from sample to sample, it is necessary to normalize the measured levels of the RNA of interest to a suitable reference RNA, but given the month-long duration of the starvation period, no endogenous RNAs could be expected to remain at the same level throughout the experiment. A whole-cell spike-in method is useful for such circumstances (Gummesson et al 2020) and is carried out by spiking each experimental sample with a small fixed relative volume of reference bacterial cells, followed by normalization of the level of the RNA of interest to one or more RNA species that are highly abundant in the spike-in cells and absent in the experimental samples. We used a spike-in culture of E. coli that overexpresses the rare transfer RNA tRNA selC , which is undetectable in the experimental samples (Svenningsen et al 2017.…”

Bacterial Chromosome Replication and DNA Repair During the Stringent Response

“…Accumulation of (p)ppGpp rapidly alters the levels of a wide range of gene transcripts and metabolites to allow cell survival and adaptation to new growth conditions ( Eymann et al, 2002 ; Traxler et al, 2008 ). The major changes involve transcriptional down-regulation of genes involved in stable RNA (rRNA and tRNA) synthesis, DNA replication, and metabolic pathways, whereas genes engaged in stress and amino-acid biosynthesis are activated ( Sanchez-Vazquez et al, 2019 ; Gummesson et al, 2020 ). In E. coli , (p)ppGpp directly binds two sites on RNA polymerase (RNAP) to allosterically alter its binding to- and efficiency at different gene promoters, which results in genome-wide transcriptional reprogramming.…”