Magnesium regulates transcription of the mgtA (magnesium transporter) gene in Salmonella enterica serovar typhimurium via prolyl-bond formation during translation of the mgtL leader ORF

In Enterobacteriaceae, the mgtA gene encodes a P-type ATPase that mediates Mg2+ uptake and is up-regulated by the PhoQP two-component system during invasion of host epithelial cells and macrophages. The regulation of mgtA has recently gained special interest given that it exists at several stages, including transcription, post-transcription and post-translation, in response to Mg2+availability. The mgtA mRNA has a 264-nucleotide 5’ leader that contains a 17 codon, proline-rich ORF, termed mgtL, whose translation has been proposed to affect the folding of the 5’ leader mRNA, which in turn regulates whether transcription is terminated before the mgtA structural gene at high Mg2+ concentrations, or is allowed to read through at limiting Mg2+ concentrations. We hypothesize that Mg2+ directly regulates translation of mgtL by facilitating prolyl-bond formation. We find that rescue of ribosome stalling at proline codons of mgtL by translation factor EF-P and methylation of tRNAPro with m1G37 by TrmD both play roles in the regulation of mgtLtranslation. Of potential significance is that TrmD is dependent on Mg2+ for its tRNA methylation activity, implying an underlying role of Mg 2+ in the regulation. We suggest a complex interaction between Mg 2+, proline, EF-P and TrmD in the regulation of mgtL translation and mgtA transcription. In addition, we provide preliminary results implicating an unknown transcription factor and other potential growth conditions in the regulation of mgtA expression

The role of the stringent response and Spx in stress response and thermotolerance development

All organisms are frequently exposed to changing environmental conditions in their natural habitat, which can impose stress and threatens basic biological functions. To cope with such adverse conditions, cells have developed complex stress response systems which allow the sensing and integration of envi- ronmental stress signals and the regulation of appropriate responses. For example, a sudden temperature upshift and other proteotoxic conditions which facilitate the misfolding and aggregation of cellular pro- teins, activate the heat shock response. This global transcriptional response mediates a concerted up- regulation and accumulation of conserved chaperones and proteases, the protein quality control system, to restore and maintain protein homeostasis during stress. Another fast-acting bacterial stress response program, the stringent response, is activated upon amino acid starvation and many other stress signals. It is regulated by the second messenger nucleotide (p)ppGpp, which mediates the transcriptional repres- sion of ribosomal genes while activating stress response- and amino acid synthesis genes, but also causes the inhibition of translation, replication and interferes with other processes. In this thesis, the heat shock response of the Gram positive model organism Bacillus subtilis was studied. It could be demonstrated that the transcriptional regulator Spx is not only a central regulator of many heat shock response genes, but can also participate in the transcriptional down-regulation of rRNA and ribosomal protein genes, which were observed to be strongly down-regulated during many stress conditions. In addition, it could be demonstrated, that the stringent response mediated by (p)ppGpp is activated during the heat shock response. Increased (p)ppGpp levels conferred elevated heat stress re- sistance while the lack of (p)ppGpp renders cells more sensitive to stress. Remarkably, it appears that both (p)ppGpp and Spx are concurrently involved in the down-regulation of rRNA genes during heat stress. Furthermore, the results suggest that (p)ppGpp is involved in direct adjustments of translation during stress, which appears to be crucial for the protective role of the stringent response in the heat stress response. Together, the results suggest a model by which the heat shock response of B. subtilis not only involves the synthesis and accumulation of chaperones and proteases of the protein quality control system but also the concurrent curbing of the protein synthesis rate by (p)ppGpp to support protein homeostasis by reducing the load for the cellular protein quality control system

REGULATION OF TRANSLATION AND TRANSCRIPTION BY SIRT1: POTENTIAL NOVEL MECHANISMS FOR REGULATING STRESS RESPONSE AND AGING

SIRT1 is a NAD+ dependent deacetylase that targets many histone and non histone proteins, thereby regulating a broad range of physiological processes such as metabolism, reproduction, development, and cell survival. In this study, we have identified novel binding partners for SIRT1 and documented three different cellular processes that are affected by these novel interactions.Using a yeast two-hybrid screen, we have identified several potential binding partners for SIRT1. Transducin like Enhancer of split 1 (TLE1) and eIF2-alpha (eIF2) are two such proteins identified in the screen whose interaction with SIRT1 was further confirmed by co-immunoprecipitation. TLE1 is co-repressor for several transcriptional factors including NF-κB. We demonstrate that SIRT1 and TLE1 repress NF-κB activity and that the catalytic activity of SIRT1 may not be critical for this. Using knock-out cell lines, we further demonstrate that both SIRT1 and TLE1 are required for the down-regulation of NF-κB activity. Our results suggest that the interaction between SIRT1 and TLE1 is important for mediating repression of NF-κB activity, potentially through a deacetyalse independent mechanism. SIRT1 protects cells from genetoxic and oxidative stress, whereas phosphorylation of eIF2 is critical for translation attenuation and preferential expression of stress related genes under stress conditions. We demonstrate that SIRT1 depleted cells show higher levels of phosphorylated eIF2 and delayed expression of the stress response protein, CHOP. Furthermore, SIRT1 deficient cells show higher sensitivity to stress treatments and a delayed recovery of protein systhesis. SIRT1 associates with eIF2 regardless of stress condition, SIRT1's catalytic activity or the phosphorylation state of eIF2. These observations suggest a novel aspect of SIRT1 mediated regulation of cellular stress response.Both SIRT1 and the target of rapamycin (TOR) are involved in age related diseases and lifespan. We demonstrate for the first time that these two pathways are interconnected. We show that SIRT1 null mouse embryonic fibroblasts (MEFs) have larger cell morphology and upregulated mTOR signaling. Furthermore, SIRT1 activator reduces, whereas inhibitor activates the mTOR pathway. Rapamycin is effective in inhibiting mTOR activity in both SIRT1 positive and deficient cells. Finally, we show that SIRT1 physically associates with TSC2 in HeLa cells. These observations demonstrate that SIRT1 negatively regulates mTOR pathway upstream of mTOR complex-1 (TORC1), potentially, by regulating the TSC1/2 complex

Environmental Perturbations Lift the Degeneracy of the Genetic Code to Regulate Protein Levels in Bacteria

The genetic code underlying protein synthesis is a canonical example of a degenerate biological system. Degeneracies in physical and biological systems can be lifted by external perturbations, thus allowing degenerate systems to exhibit a wide range of behaviors. Here we show that the degeneracy of the genetic code is lifted by environmental perturbations to regulate protein levels in living cells. By measuring protein synthesis rates from a synthetic reporter library in Escherichia coli, we find that environmental perturbations, such as reduction of cognate amino acid supply, lift the degeneracy of the genetic code by splitting codon families into a hierarchy of robust and sensitive synonymous codons. Rates of protein synthesis associated with robust codons are up to 100-fold higher than those associated with sensitive codons under these conditions. We find that the observed hierarchy between synonymous codons is not determined by usual rules associated with tRNA abundance and codon usage. Rather, competition among tRNA isoacceptors for aminoacylation underlies the robustness of protein synthesis. Remarkably, the hierarchy established using the synthetic library also explains the measured robustness of synthesis for endogenous proteins in E. coli. We further found that the same hierarchy is reflected in the fitness cost of synonymous mutations in amino acid biosynthesis genes and in the transcriptional control of σ-factor genes. Our study suggests that organisms can exploit degeneracy lifting as a general strategy to adapt protein synthesis to their environment.Molecular and Cellular Biolog

Emergence of robust growth laws from optimal regulation of ribosome synthesis

Bacteria must constantly adapt their growth to changes in nutrient availability; yet despite large‐scale changes in protein expression associated with sensing, adaptation, and processing different environmental nutrients, simple growth laws connect the ribosome abundance and the growth rate. Here, we investigate the origin of these growth laws by analyzing the features of ribosomal regulation that coordinate proteome‐wide expression changes with cell growth in a variety of nutrient conditions in the model organism Escherichia coli. We identify supply‐driven feedforward activation of ribosomal protein synthesis as the key regulatory motif maximizing amino acid flux, and autonomously guiding a cell to achieve optimal growth in different environments. The growth laws emerge naturally from the robust regulatory strategy underlying growth rate control, irrespective of the details of the molecular implementation. The study highlights the interplay between phenomenological modeling and molecular mechanisms in uncovering fundamental operating constraints, with implications for endogenous and synthetic design of microorganisms.ISSN:1744-429

Mechanism of Gene Regulation by Coding PolyA Tracks

Regulation of gene expression is essential for cellular development and survival. The great variety and complexity of regulatory mechanisms underscores this fact. Messenger RNA stability and translational efficiency are often key determinants of gene expression. mRNA surveillance pathways, discovered for their role in degradation of aberrant mRNA, are now known to be instrumental in the regulation of physiologically correct mRNA stability. Thus, the study of cis elements in a transcript that can induce mRNA surveillance pathways has become an area of particular interest. Here I report on the mechanism of gene regulation by coding polyA tracks, defined as a sequence of at least 12 consecutive nucleotides in which all but one are adenosines. When a polyA track is present in the open reading frame of an mRNA, the translating ribosome stalls and frameshifts due to interactions with the polyA sequence. These events lead to degradation of the transcripts by the mRNA surveillance pathways no-go decay and nonsense mediated decay. As a consequence of the polyA sequence, less protein is expressed from these transcripts. Approximately 2% of genes in most genomes, including humans, contain a polyA track and are potentially regulated by this mechanism

Regulación de las metiltransferasas del ARNr 16S de resistencia a aminoglucósidos: análisis funcional y mutacional del 5’ UTR de armA

Tesis de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 27/09/2017Antimicrobial resistance remains one of the most serious threats to modern medicine. As such, the selection and administration of antibiotics must be carefully considered, especially since the discovery of novel antimicrobial agents has reduced substantially. Agents that have previously been dismissed for unfavorable secondary activity are becoming increasingly valuable as the resistance prevalence towards the first choice antibiotic increases drastically. The aminoglycosides are a family of antibiotics that belong to this category. Shortly after their introduction to the market, problems with toxicity became apparent and they were no longer considered a drug of choice for physicians. However, increasingly worrisome resistance trends have forced physicians to reevaluate the application of aminoglycosides for life-threatening infections caused by Gram-negative pathogens. Among other resistance mechanisms, the 16S ribosomal RNA methyltransferases have emerged in Gram-negative pathogenic bacteria as an acquired resistance mechanism conferring high-level resistance to all clinically relevant aminoglycosides, even ones that have not yet been released to the market. Since their discovery in 2003, a total of 13 acquired methyltransferases have been identified including three variants. The rate at which these resistance determinants are spreading, combined with the broad resistance profile they confer to most clinically significant aminoglycosides, is jeopardizing the aminoglycosides as a viable last resort antibiotic. Initially, the objectives of the here presented doctoral thesis were to perform a comprehensive in silico analysis of the various families of 16S rRNA methyltransferases, including acquired resistance conferring-, intrinsic resistance conferring- and housekeeping- methyltransferases. The aim of this was to identify a putative origin of the acquired resistance conferring methyltransferases, to potentially discovering their ancestral regulatory mechanism. This study revealed that the acquired 16S methyltransferases most likely convergently evolved from a number of different RNA methyltransferases. Furthermore, this study also demonstrated a high degree of plasticity in the 5’ upstream regions between the various acquired resistance conferring methyltransferases, although the region immediately upstream of the coding region was relatively conserved throughout isolate of the same genes. This plasticity is most likely a result of the highly versatile mobile genetic elements in which these acquired resistance determinants are found...La resistencia a antibióticos supone una de las principales amenazas para la medicina moderna. Esto, junto con la drástica disminución del descubrimiento de nuevas moléculas antibióticas, hace que la elección y administración de tratamientos antibióticos deba ser controlada escrupulosamente. Fármacos que anteriormente eran rechazados por sus efectos adversos están comenzando a utilizarse de nuevo debido a los elevados niveles de resistencia a los antibióticos de primera elección. Los aminoglucósidos son un grupo de antibióticos que pertenecen a esta categoría. Poco después de su introducción en el mercado fueron desechados como fármacos de elección debido a su elevada toxicidad. Sin embargo, el alarmante incremento de la resistencia a antibióticos ha obligado a los clínicos a reevaluar el uso de los aminoglucósidos para tratar infecciones graves producidas por patógenos Gram negativos. Entre otros mecanismos de resistencia, las metiltransferasas del ARNr 16S han emergido en bacterias patógenas Gram negativas como un mecanismo de resistencia adquirido, confiriendo elevados niveles de resistencia frente a todos los aminoglucósidos clínicamente relevantes, incluso también frente a nuevos aminoglucósidos que todavía no han salido al mercado. Desde su descubrimiento en el año 2003, se han identificado un total de 13 metiltransferasas, incluyendo tres variantes. La velocidad a la que estos determinantes de resistencia se están diseminando, junto con el amplio espectro de aminoglucósidos frente a los que confieren resistencia, está poniendo en peligro el empleo de este grupo de fármacos como antibióticos de último recurso. Inicialmente, los objetivos de esta Tesis Doctoral consistieron en realizar un estudio in silico en profundidad de las diferentes familias de metiltransferasas del ARNr 16S, que incluyen a las metiltransferasas de resistencia, tanto adquiridas como intrínsecas, y a las metiltransferasas endógenas. El objetivo de este análisis fue poder identificar el origen putativo de las metiltransferasas de resistencia adquiridas, con la finalidad de descubrir el origen de su mecanismo de regulación. Este estudio reveló que las metiltransferasas adquiridas del ARNr 16S probablemente evolucionaron de manera convergente a partir de las metilasas intrínsecas del ARN. Además, este análisis también mostró el alto grado de plasticidad en la región 5´ no codificante de las metiltransferasas adquiridas, aunque en distintos aislados de cada uno de los genes codificantes pudimos observar que la región inmediatamente corriente arriba de la secuencia codificante presentaba un alto grado de conservación. Esta plasticidad es debida, presumiblemente, a la versatilidad de los elementos genéticos móviles en los que estos determinantes de resistencia se localizan...Depto. de Sanidad AnimalFac. de VeterinariaTRUEunpu