Short‐term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate

Abstract

Ribosomes are absolutely essential for growth but are, moreover, energetically costly to produce. Therefore, it is important to adjust the cellular ribosome levels according to the environmental conditions in order to obtain the highest possible growth rate while avoiding energy wastage on excess ribosome biosynthesis. Here we show, by three different methods, that the ribosomal RNA content of Escherichia coli is downregulated within minutes of the removal of an essential nutrient from the growth medium, or after transcription initiation is inhibited. The kinetics of the ribosomal RNA reduction vary depending on which nutrient the cells are starved for. The number of ribosomes per OD unit of cells is roughly halved after 80 min of starvation for isoleucine or phosphate, while the ribosome reduction is less extensive when the cells are starved for glucose. Collectively, the results presented here support the simple model proposed previously, which identifies the inactive ribosomal subunits as the substrates for degradation, since the most substantial rRNA degradation is observed under the starvation conditions that most directly affect the protein synthesis. K E Y W O R D Sbacterial stress response, Escherichia coli, nutrient starvation, ribosomal RNA, stable RNA degradation

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

“…The methodology allowed us to quantify the changes in total rRNA and total mRNA per OD unit of bacterial culture over the first 80 min of starvation for isoleucine. In accordance with other reports (Ben-Hamida and Schlessinger, 1966;Jacobson and Gillespie, 1968;Maruyama and Mizuno, 1970;Zundel et al, 2009;Piir et al, 2011;Fessler et al, 2020), we find that the stability of rRNA is compromised upon nutrient starvation, resulting in a drop in rRNA per OD unit to 70% of the pre-starvation level within the first 80 min of an amino acid starvation. Because rRNA constitutes the vast majority of cellular RNA, this drop affects the quantification of all other RNA species in the cell if the RNAseq data is normalized solely to the sequencing depth of the samples in the conventional way (referred to here as RPKM-normalization, Figure 6).…”

“…As seen in Supplementary Figure S1A, the ratio of plasmid reads to total reads of the three replicates taken during steady-state growth varied only by~1%, indicating a high reproducibility of the data. In contrast, as starvation progressed within the 80-min time series, the ratio of plasmid reads/total reads increased, indicative of a decline in total RNA levels from the experimental samples, which is consistent with the net negative effect of ppGpp on the activity of RNA polymerase (Fiil et al, 1972;Sarubbi et al, 1988) and breakdown of rRNA (Zundel et al, 2009;Fessler et al, 2020) and tRNA (Svenningsen et al, 2017) upon starvation. However, the correlation deviated from the expected ratio at the 20-min time point with approximately 30% from the trend.…”

“…However, while rRNA and tRNAs are generally believed to be stable during exponential growth (Baracchini and Bremer, 1987), the familiar way of thinking of these RNAs as stable in an absolute sense has been questioned for some time (Deutscher, 2003). Our previous work shows that a substantial fraction of the tRNA and rRNA pools in the cell is rapidly degraded upon amino acid starvation (Svenningsen et al, 2017;Fessler et al, 2020), suggesting that the total RNA content of E. coli cells may decrease appreciably under this condition. Given the global changes in gene expression and the possibility that total RNA levels may decrease upon amino acid starvation we reasoned that a normalization method that is independent of any assumptions about cellular RNA content would be important for accurate detection of gene expression changes during the stringent response.…”

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

“…Unstable rRNA has been also reported, using a similar normalization method than with tRNA studies. Fessler et al (2020) looked at the kinetics of different filter-resuspend starvation regimens, showing that while glucose starvation produces minimal instability of rRNA, isoleucine and phosphate starvation reduced ribosome content in a similar manner than previosly observed by tRNA ( Svenningsen et al, 2016 ). Again, the authors concluded that although (p)ppGpp may contribute, it is not essential for the degradation of rRNA, instead, inactive ribosomes are vulnerable to degradation.…”

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

“…While changes in OD provide a clear measure of the growth of bacterial cultures in the steady state, where all cell constituents grow with the same rate, changes in optical density are complex to interpret upon disruption of the steady state, e.g. by starvation (Fessler et al 2020, Koch 1970. Accordingly, the correlation between OD measurements and bacterial concentration (CFU/ml) was good for exponentially growing cultures, but poor for the starved cultures ( Supplementary Fig.S1) .…”