Abstract:26Bacteria initially respond to conditions that attenuate their growth by eliciting large-scale 27 transcriptional changes. The accompanying changes in gene expression and metabolism allow 28 the bacterial cells to effectively adapt to the growth attenuated state. How the transcriptome 29 subsequently changes as growth attenuation ensues is not well understood. We used nitrogen 30 (N) starvation as a model nutrient starvation condition to study the transcriptome of growth 31 attenuated Escherichia coli. The re… Show more
“…It is well established that rRNA becomes degraded in both N‐ and carbon (C)‐starved E. coli , and this concomitantly results in a decrease in the cellular ribosome content (Dai et al., 2016; Li et al., 2018). Consistent with this, we recently reported that 16S and 23S rRNAs are gradually degraded over the initial 24 hr under N starvation, that is, over a period of time that coincides with H‐body formation (McQuail et al., 2020; Switzer et al., 2020). RNase E is an important component in the rRNA degradation pathway in E. coli (Sulthana et al., 2016).…”
Section: Resultssupporting
confidence: 70%
“…Recently we reported that, despite running out of N, E. coli cells remain transcriptionally active over the initial 24 hr under N starvation (Switzer et al., 2020), with ~10% of the transcriptome differentially expressed (242 genes upregulated and 209 genes downregulated). Rifampicin treatment immediately following N run‐out had a detrimental effect on cell viability at N‐24 (~50% reduction compared to untreated) suggesting that the transcriptional activity during the initial 24 hr following N run‐out is important for optimal cell viability (Switzer et al., 2020). Rifampicin treatment will lead to the inhibition of de novo transcription and subsequent depletion of the cellular RNA pool over time.…”
Under conditions of nutrient adversity, bacteria adjust metabolism to minimize cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualized together in live bacteria. We now show that in long‐term nitrogen starved E. coli, both RNase E and Hfq co‐localize in a single, large focus. This subcellular assembly, which we refer to as the H‐body, forms by a liquid‐liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of a hitherto unreported subcellular compartmentalization of a process(s) associated with RNA management in stressed bacteria.
“…It is well established that rRNA becomes degraded in both N‐ and carbon (C)‐starved E. coli , and this concomitantly results in a decrease in the cellular ribosome content (Dai et al., 2016; Li et al., 2018). Consistent with this, we recently reported that 16S and 23S rRNAs are gradually degraded over the initial 24 hr under N starvation, that is, over a period of time that coincides with H‐body formation (McQuail et al., 2020; Switzer et al., 2020). RNase E is an important component in the rRNA degradation pathway in E. coli (Sulthana et al., 2016).…”
Section: Resultssupporting
confidence: 70%
“…Recently we reported that, despite running out of N, E. coli cells remain transcriptionally active over the initial 24 hr under N starvation (Switzer et al., 2020), with ~10% of the transcriptome differentially expressed (242 genes upregulated and 209 genes downregulated). Rifampicin treatment immediately following N run‐out had a detrimental effect on cell viability at N‐24 (~50% reduction compared to untreated) suggesting that the transcriptional activity during the initial 24 hr following N run‐out is important for optimal cell viability (Switzer et al., 2020). Rifampicin treatment will lead to the inhibition of de novo transcription and subsequent depletion of the cellular RNA pool over time.…”
Under conditions of nutrient adversity, bacteria adjust metabolism to minimize cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualized together in live bacteria. We now show that in long‐term nitrogen starved E. coli, both RNase E and Hfq co‐localize in a single, large focus. This subcellular assembly, which we refer to as the H‐body, forms by a liquid‐liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of a hitherto unreported subcellular compartmentalization of a process(s) associated with RNA management in stressed bacteria.
“…The error bars represent ± SD. Note: although no nitrogen source was present in the medium, some minor growth (especially of the control and wild‐type strains) remained (A), which is a known phenomenon within the first hours of cultivation after nitrogen depletion (Switzer et al , 2020). Source data are available online for this figure.…”
One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.
“…Moreover, previous studies on ntrC in E. coli, showed interplay between NtrC and adaptation to nitrogen starvation. These studies were conducted in short term nitrogen starvation, from 20 minutes to 34-days (15,31,32). But, there is no study reported on the survival of ∆ntrC knockout mutant in long term nutrient starvation.…”
Section: The Role Of Ntrc In Long Term Starvationmentioning
confidence: 99%
“…Consequently, ∆ntrC knockout mutant died after 150 days of nitrogen starvation. Even, Switzer et al have shown that, inhibition of transcription at the onset nitrogen starvation has detrimental effect nitrogen starvation survival ability of E. coli (31). Therefore, NtrC which is a transcriptional activator of nitrogen transporter and metabolism genes, is essential for cell survival in long term nitrogen starvation.…”
Section: The Role Of Ntrc In Long Term Starvationmentioning
The enteric pathogens cycle between nutrient rich host and nutrient poor external environment. These pathogens compete for nutrients in the host as well as in external environment and also often experience starvation. In this context, we have studied the role of a global nitrogen regulator (NtrC) of Salmonella Typhimurium. The ntrC mutation caused extended lag phase and slow growth in minimal media. In lag phase the wild-type cells showed ~60 fold more expression of ntrC as compared to log phase cells. The role of ntrC gene is often studied with respect to nitrogen scavenging in a low nitrogen growing condition. However, our observation indicates that, even in the adequate supply of nitrogen, the ntrC null mutants failed to adapt to new nutrient conditions and were slow to exit from the lag phase. Gene expression studies at lag phase showed down-regulation of the genes involved in carbon/nitrogen transportation and metabolism in ?ntrC mutant. In the co-culture competition studies, we observed that ntrC knockout was unable to survive with the wild-type Salmonella and E. coli. We also observed that the ΔntrC mutant did not survive long-term nitrogen starvation (150 days). Critical analysis of starvation survival revealed that ntrC gene is essential for recycling of nutrients from the dead cells. The nutrient recycling efficiency of ?ntrC mutant was ~ 12 times less efficient than wild-type. Hence, ntrC expression at the lag phase is essential for the competitive fitness of Salmonella to survive in an environment having low and fluctuating nutrient conditions.
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