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Non-canonical Amino Acid Substrates of E. coli Aminoacyl-tRNA Synthetases.
Hartman, MCT
Chembiochem : a European journal of chemical biology. 2022;(1):e202100299
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Abstract
In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.
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Escherichia coli small molecule metabolism at the host-microorganism interface.
Gatsios, A, Kim, CS, Crawford, JM
Nature chemical biology. 2021;(10):1016-1026
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Abstract
Escherichia coli are a common component of the human microbiota, and isolates exhibit probiotic, commensal and pathogenic roles in the host. E. coli members often use diverse small molecule chemistry to regulate intrabacterial, intermicrobial and host-bacterial interactions. While E. coli are considered to be a well-studied model organism in biology, much of their chemical arsenal has only more recently been defined, and much remains to be explored. Here we describe chemical signaling systems in E. coli in the context of the broader field of metabolism at the host-bacteria interface and the role of this signaling in disease modulation.
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Studying bacterial chemosensory array with CryoEM.
Qin, Z, Zhang, P
Biochemical Society transactions. 2021;(5):2081-2089
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Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.
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The Classical, Yet Controversial, First Enzyme of Lipid Synthesis: Escherichia coli Acetyl-CoA Carboxylase.
Cronan, JE
Microbiology and molecular biology reviews : MMBR. 2021;(3):e0003221
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Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA, the building block of fatty acid synthesis, is the paradigm bacterial ACC. Many reports on the structures and stoichiometry of the four subunits comprising the active enzyme as well as on regulation of ACC activity and expression have appeared in the almost 20 years since this subject was last reviewed. This review seeks to update and expand on these reports.
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Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis.
Smolskaya, S, Logashina, YA, Andreev, YA
International journal of molecular sciences. 2020;(3)
Abstract
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
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6.
Copper tolerance in bacteria requires the activation of multiple accessory pathways.
Giachino, A, Waldron, KJ
Molecular microbiology. 2020;(3):377-390
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Copper is a required micronutrient for bacteria and an essential cofactor for redox-active cuproenzymes. Yet, excess copper is extremely toxic, and is exploited as a bacteriocide in medical and biotechnological applications and also by the mammalian immune system. To evade copper toxicity, bacteria not only control intracellular copper homeostasis, but they must also repair the damage caused by excess copper. In this review, we summarize the bacterial cell-wide response to copper toxicity in Enterobacteria. Tapping into the abundant research data on two key organisms, Escherichia coli and Salmonella enterica, we show that copper resistance requires both the direct copper homeostatic response and also the indirect accessory pathways that deal with copper-induced damage. Since patterns of copper response are conserved through the Proteobacteria, we propose a cell-wide view of copper detoxification and copper tolerance that can be used to identify novel targets for copper-based antibacterial therapeutics.
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Protein Disulfide Exchange by the Intramembrane Enzymes DsbB, DsbD, and CcdA.
Bushweller, JH
Journal of molecular biology. 2020;(18):5091-5103
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Abstract
The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.
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The SUF system: an ABC ATPase-dependent protein complex with a role in Fe-S cluster biogenesis.
Garcia, PS, Gribaldo, S, Py, B, Barras, F
Research in microbiology. 2019;(8):426-434
Abstract
Iron-sulfur (Fe-S) clusters are considered one of the most ancient and versatile inorganic cofactors present in the three domains of life. Fe-S clusters can act as redox sensors or catalysts and are found to be used by a large number of functional and structurally diverse proteins. Here, we cover current knowledge of the SUF multiprotein machinery that synthesizes and inserts Fe-S clusters into proteins. Specific focus is put on the ABC ATPase SufC, which contributes to building Fe-S clusters, and appeared early on during evolution.
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Control of the phoBR Regulon in Escherichia coli.
Gardner, SG, McCleary, WR
EcoSal Plus. 2019;(2)
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Abstract
Phosphorus is required for many biological molecules and essential functions, including DNA replication, transcription of RNA, protein translation, posttranslational modifications, and numerous facets of metabolism. In order to maintain the proper level of phosphate for these processes, many bacteria adapt to changes in environmental phosphate levels. The mechanisms for sensing phosphate levels and adapting to changes have been extensively studied for multiple organisms. The phosphate response of Escherichia coli alters the expression of numerous genes, many of which are involved in the acquisition and scavenging of phosphate more efficiently. This review shares findings on the mechanisms by which E. coli cells sense and respond to changes in environmental inorganic phosphate concentrations by reviewing the genes and proteins that regulate this response. The PhoR/PhoB two-component signal transduction system is central to this process and works in association with the high-affinity phosphate transporter encoded by the pstSCAB genes and the PhoU protein. Multiple models to explain how this process is regulated are discussed.
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10.
When size matters - coordination of growth and cell cycle in bacteria.
Morcinek-Orłowska, J, Galińska, J, Glinkowska, MK
Acta biochimica Polonica. 2019;(2):139-146
Abstract
Bacterial cells often inhabit environments where conditions can change rapidly. Therefore, a lot of bacterial species developed control strategies allowing them to grow and divide very fast during feast and slow down both parameters during famine. Under rich nutritional conditions, fast-growing bacteria can divide with time interval equal to half of the period required to synthesize their chromosomes. This is possible due to multifork replication which allows ancestor cells to start copying genetic material for their descendants. This reproduction scheme was most likely selected for, since it enables maximization of growth rate and hence - effective competition for resources, while ensuring that DNA replication will not become limiting for cell division. Even with this complexity of cell cycle, isogenic bacterial cells grown under defined conditions display remarkably narrow distribution of sizes. This may suggest that mechanisms exists to control cell size at division step. Alternative view, with great support in experimental data is that the only step coordinated with cell growth is the initiation of DNA replication. Despite decades of research we are still not sure what the driving forces in bacterial cell cycle are. In this work we review recent advances in understanding coordination of growth with DNA replication coming from single cell studies and systems biology approaches.