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A multistep enrichment process with custom growth medium improves resuscitation of chlorine-stressed coliforms from secondary sewage effluents.
Mobberley, JM, Cooley, SK, Widder, MW, Phillips, SM, Melville, AM, Brennan, LM, Divito, VT, van der Schalie, WH, Ozanich, RM, Hutchison, JR
Journal of microbiological methods. 2022;:106364
Abstract
Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study, we report that the addition of a growth enhancer mix consisting of trehalose, sodium pyruvate, magnesium chloride, and 1× trace mineral supplement improved growth of microorganisms from chlorinated secondary effluent in the base medium with Colilert-18. Improving growth of chlorine stressed microorganisms from secondary effluent is crucial to decreased detection time from 18 to 8 h.
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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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Equilibrium properties of E. coli lactose permease symport-A random-walk model approach.
Sun, H
PloS one. 2022;(2):e0263286
Abstract
The symport of lactose and H+ is an important physiological process in E. coli, for it is closely related to cellular energy supply. In this paper, we review, extend and analyse a newly proposed cotransport model that takes the "leakage" phenomenon (uncoupled particle translocation) into account and also satisfies the static head equilibrium condition. Then, we use the model to study the equilibrium properties, including equilibrium solution and the time required to reach equilibrium, of the symport process of E. coli LacY protein, when varying the parameters of the initial state of cotransport system. It can be found that in our extended model, H+ and lactose will reach their equilibrium state separately, and when "leakage" exists, it linearly affects the equilibrium solution, which is a useful property that the original model does not have. We later investigated the effect of the volume of periplasm and cytoplasm on the equilibrium properties. For a certain E. coli cell, as it continues to lose water and contract, the time for cytoplasm pH to be stabilized by symport increases monotonically when the cell survives. Finally, we reproduce the experimental data from a literature to verify the validity of the extension in this symport process. The above phenomena and other findings in this paper may help us to not only further validate or improve the model, but also deepen our understanding of the cotransport process of E. coli LacY protein.
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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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Discovery of an Unnatural DNA Modification Derived from a Natural Secondary Metabolite.
Wang, T, Kohli, RM
Cell chemical biology. 2021;(1):97-104.e4
Abstract
Despite widespread interest for understanding how modified bases have evolved their contemporary functions, limited experimental evidence exists for measuring how close an organism is to accidentally creating a new, modified base within the framework of its existing genome. Here, we describe the biochemical and structural basis for how a single-point mutation in E. coli's naturally occurring cytosine methyltransferase can surprisingly endow a neomorphic ability to create the unnatural DNA base, 5-carboxymethylcytosine (5cxmC), in vivo. Mass spectrometry, bacterial genetics, and structure-guided biochemistry reveal this base to be exclusively derived from the natural but sparse secondary metabolite carboxy-S-adenosyl-L-methionine (CxSAM). Our discovery of a new, unnatural DNA modification reveals insights into the substrate selectivity of DNA methyltransferase enzymes, offers a promising new biotechnological tool for the characterization of the mammalian epigenome, and provides an unexpected model for how neomorphic bases could arise in nature from repurposed host metabolites.
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An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions.
Somma, V, Calvio, C, Rabuffetti, M, Rama, E, Speranza, G, Morelli, CF
Bioorganic chemistry. 2021;:105217
Abstract
γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.
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In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code.
Kugele, A, Ketter, S, Silkenath, B, Wittmann, V, Joseph, B, Drescher, M
Chemical communications (Cambridge, England). 2021;(96):12980-12983
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Abstract
The membrane transporter BtuB is site-directedly spin labelled on the surface of living Escherichia coli via Diels-Alder click chemistry of the genetically encoded amino acid SCO-L-lysine. The previously introduced photoactivatable nitroxide PaNDA prevents off-target labelling, is used for distance measurements, and the temporally shifted activation of the nitroxide allows for advanced experimental setups. This study describes significant evolution of Diels-Alder-mediated spin labelling on cellular surfaces and opens up new vistas for the the study of membrane proteins.
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Gender medicine: the impact of probiotics on male patients.
Pacifici, L, Santacroce, L, Dipalma, G, Haxhirexha, K, Topi, S, Cantore, S, Altini, V, Pacifici, A, De Vito, D, Pettini, F, et al
La Clinica terapeutica. 2021;(1):e8-e15
Abstract
PURPOSE Numerous studies in the literature confirm the importance of the use of probiotics in inflammatory states of the prostate in humans. Our pilot study aimed to test probiotics strains ability to improve urinary bacterial load in male subjects. METHODS Twenty healthy men aged 55-65 years, with recurrent urinatory infections were enrolled and randomized into 2 groups. Urine culturing to detect Enterobacteriaceae (Escherichia Coli and Enterococcus faecalis) was performed at baseline and at 12 weeks after the starting of the study. RESULTS We found that taking the probiotic reduces the bacterial load of E. coli and E. faecalis in urine cultures. The use of the probiotic in these inflammatory states does not affect the change in weight in the tested subjects. CONCLUSIONS Our data confirm and improve the main results reported in the scientific literature on the importance of taking probiotics in prostatitis.
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Protein Mass-Modulated Effects in Alkaline Phosphatase.
Ghosh, AK, Schramm, VL
Biochemistry. 2021;(2):118-124
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Recent experimental studies engaging isotopically substituted protein (heavy protein) have revealed that many, but not all, enzymatic systems exhibit altered chemical steps in response to an altered mass. The results have been interpreted as femtosecond protein dynamics at the active site being linked (or not) to transition-state barrier crossing. An altered enzyme mass can influence several kinetic parameters (kcat, Km, and kchem) in amounts of ≤30% relative to light enzymes. An early report on deuterium-labeled Escherichia coli alkaline phosphatase (AP) showed an unusually large enzyme kinetic isotope effect on kcat. We examined steady-state and chemical step properties of native AP, [2H]AP, and [2H,13C,15N]AP to characterize the role of heavy enzyme protein dynamics in reactions catalyzed by AP. Both [2H]- and [2H,13C,15N]APs showed unaltered steady-state and single-turnover rate constants. These findings characterize AP as one of the enzymes in which mass-dependent catalytic site dynamics is dominated by reactant-linked atomic motions. Two catalytic site zinc ions activate the oxygen nucleophiles in the catalytic site of AP. The mass of the zinc ions is unchanged in light and heavy APs. They are essentially linked to catalysis and provide a possible explanation for the loss of linkage between catalysis and protein mass in these enzymes.
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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.