Due to the success of the Research Topic <a href="https://www.frontiersin.org/research-topics/12842/interview-with-the-translational-apparatus-stories-of-intriguing-circuits-and-mechanisms-to-regulate">Interview with the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria</a> we are launching a volume II of the collection.

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Frontiers in Microbiology

Microbial Physiology and Metabolism

Interview with the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria. Vol II

Bacteria are continuously subjected to a variety of stressors, including fluctuations in temperature, pH, nutrient availability, salt and oxygen concentration, which can cause genetic reprogramming and alteration in cell metabolism, growth and survival. By modulating protein levels, translational control provides a swift and accurate response to cope with and react rapidly to these diverse stressors.In the previous Research Topic of “Interview with the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria” we compiled several manuscripts that highlighted the molecular diversity of bacterial translation regulation and the various techniques employed to study it (<a href="#B5">Giuliodori and Marzi, 2021</a>). For Volume II, we have included four additional original research articles that examine the regulation of translation in Escherichia coli under stressful conditions such as cold acclimatization, oxidative stress, and variations of intracellular phosphate concentration. These articles identify and describe the different elements that play a role in regulating gene expression at the translational level in one of the most extensively studied model organisms. Understanding the mechanisms by which bacteria respond to stress can help to better understand their biology, as well as to develop new strategies for controlling bacterial populations in various environments.Enterobacteriaceae frequently encounter temperature changes as they move from the intestinal to external habitats. The sudden decrease in temperature triggers the cold-shock response, which involves an adaptation period, during which mesophilic bacteria, such as E. coli, stop growing and block or reduce the synthesis of many macromolecules. This phase sees a temporarily increase in the expression of a specific group of genes, referred to as cold-shock genes. Unlike other regulons, cold-shock gene expression is mainly regulated post-transcriptionally, either by modulating transcript half-life or by controlling translation efficiency (for a review see <a href="#B3">Giuliodori, 2016</a>; <a href="#B6">Zhang and Gross, 2021</a>).<a href="https://doi.org/10.3389/fmicb.2023.1118329">Giuliodori et al.</a> investigate the role of CspA, a key cold-shock protein in E. coli, during translation at low temperature, using molecular biology techniques such as reconstituted translation systems and probing methods. In a previous study, the authors demonstrated that the translation of cspA mRNA is facilitated by a thermosensor element that opens the ribosome binding site during cold-shock and makes it less accessible at 37°C (<a href="#B4">Giuliodori et al., 2010</a>). The present research builds upon these findings and demonstrates that CspA specifically promotes the translation of cspA mRNA folded in the conformation that is less accessible to the ribosome. The mechanism by which CspA promotes translation is by facilitating the ribosomes' progression from translation initiation to elongation (<a href="https://doi.org/10.3389/fmicb.2023.1118329">Giuliodori et al.</a>). The study suggests that this structure-dependent mechanism may also occur for other cold-shock genes targeted by CspA.Bacteria inhabiting the intestine often also experience changes in the intracellular pool of phosphate (Pi) due to dietary variations and the aforementioned transition from the intra to the extra-intestinal environment. To respond rapidly to these fluctuations, bacteria have developed a regulatory circuit consisting of a phosphate-specific transport system, encoded by the pstSCAB-phoU genes, and the PhoR-PhoB two-component system. The PhoR transmembrane sensor histidine kinase detects Pi limitation and activates PhoB, the response regulator, which controls the expression of the genes containing a Pho box (for a review see <a href="#B2">Gardner and McCleary, 2019</a>). Furthermore, Pi starvation activates the synthesis of (p)ppGpp, resulting in a decrease in rRNA and tRNA synthesis.The article from <a href="https://doi.org/10.3389/fmicb.2022.989818">Espinosa et al.</a>, investigates factors and mechanisms that downregulate protein synthesis in response to phosphate starvation in E. coli. Through the use of radiolabeling, the authors show that phosphate starvation leads to a decrease in total cellular RNA, primarily due to a reduction in rRNA, while protein synthesis continues, albeit at a reduced rate, before eventually stopping. To understand why this occurs, the authors compared the protein synthesis rate in E. coli cells before and after phosphate starvation, with and without the overproduction of an induced and long-lived mRNA that is highly translated. The data analysis suggests that the reduced amounts of mRNAs is the limiting factor for protein synthesis during phosphate starvation, which may be an adaptive response to conserve resources.Bacteria have developed defense systems against reactive oxygen species (ROS) such as superoxide (O2-) and hydrogen peroxide (H2O2) to protect their cellular components. Oxidative stress activates various stress responses, which depend on the type of ROS and microorganism (for a review see <a href="#B1">Fasnacht and Polacek, 2021</a>). In gram-negative bacteria, H2O2 typically induces the expression of the transcriptional factor OxyR, which regulates about thirty stress genes. In E. coli, an increase in O2- activates SoxR and then SoxS, which controls the transcription of about 25 genes. Alongside these regulons, oxidative stress also affects tRNA, ribosomes, and factors, resulting in a significant impact on the traditional translation process.<a href="https://doi.org/10.3389/fmicb.2022.1042675">Leiva et al.</a> reported that oxidative stress affects both translation initiation and elongation in E. coli. To examine the impact of oxidative stress on translation, the authors developed 61 GFP reporter constructs, each containing a unique sequence of four repeated amino acids inserted between the fourth and fifth codons of the GFP coding region. The system enabled the authors to demonstrate that codon choice influences GFP translation when E. coli K-12 cells are grown in minimal media, but not when they are exposed to oxidative stress. In this latter condition GFP expression is overall reduced and the rate of translation initiation influences the speed of protein synthesis more than under normal conditions.The original research carried out by <a href="https://doi.org/10.3389/fmicb.2022.1088941">Chen et al.</a> aims to explore the impact of different 5′UTR sequences on gene expression regulation in E. coli. It is known that mRNA 5′ UTR are regulatory-hubs switching on and off translation or modulating the expression of several genes. The team assessed both rate of synthesis and mRNA stability of a large collection of constructs containing synthetic 5′UTR sequences, differing at the level of the ribosome binding site and linked to various reporter genes. Their findings showed that the predicted theoretical translation initiation rate did not accurately reflect protein levels for most of the synthetic 5′UTRs. Furthermore, several 5′UTRs were found to affect mRNA stability in a reporter gene-dependent manner.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Centre National de la Recherche Scientifique (CNRS) and by the French National Research Agency ANR (ANR-21-CE12-0030-01 to SM and ANR-10-LABX-0036 NETRNA).We kindly thank all contributors, authors as well as reviewers, for their precious time, interesting ideas and input for this Research Topic published in the section of Microbial Physiology and Metabolism in Frontiers in Microbiology.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Editorial: Interview with the translational apparatus: stories of intriguing circuits and mechanisms to regulate translation in bacteria, volume II

E. coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs – including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Escherichia coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs—including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Escherichia coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. E. coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation

A set of 41 synthetic 5’UTRs with different theoretical translation initiation rates were generated to explore the role of 5’UTRs in the regulation of protein levels in Escherichia coli. The roles of the synthetic 5’UTRs in regulating the expression of different reporter genes were analyzed in vivo. Protein levels varied substantially between the different constructs but for most of the 5’UTRs, protein levels were not correlated with theoretical translation initiation rates. Large variations in mRNA concentrations were measured with the different 5’UTRs even though the same concentration of transcription inducer was used in each case. 5’UTRs were also found to strongly affect mRNA stability, and these changes in mRNA stability often contributed to observed differences in mRNA concentration. Unexpectedly, the effect of the 5’UTRs on mRNA half-lives was found to vary depending on the downstream reporter gene. These results clearly demonstrate that 5'UTRs contribute to gene expression regulation at the level of translation initiation and of mRNA stability, to an extent that depends on the nature of the downstream gene.

A set of 41 synthetic 5’UTRs with different theoretical translation initiation rates were generated to explore the role of 5’UTRs in the regulation of protein levels in Escherichia coli. The roles of the synthetic 5’UTRs in regulating the expression of different reporter genes were analyzed in vivo. Protein levels varied substantially between the different constructs but for most of the 5’UTRs, protein levels were not correlated with theoretical translation initiation rates. Large variations in mRNA concentrations were measured with the different 5’UTRs even though the same concentration of transcription inducer was used in each case. 5’UTRs were also found to strongly affect mRNA stability, and these changes in mRNA stability often contributed to observed differences in mRNA concentration. Unexpectedly, the effect of the 5’UTRs on mRNA half-lives was found to vary depending on the downstream reporter gene. These results clearly demonstrate that 5’UTRs contribute to gene expression regulation at the level of translation initiation and of mRNA stability, to an extent that depends on the nature of the downstream gene.

5’UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability

The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition. In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons. Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range. Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.

Introduction: The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition.Methods: In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons.Results: Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range.Discussion: Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.