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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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2.
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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Bioremediation of an agricultural saline soil contaminated with endosulfan and Escherichia coli by an active surface agent induced in a Penicillium crustosum culture.
Landa-Faz, A, Rodríguez-Vázquez, R, Roldán-Carrillo, TG, Hidalgo-Lara, ME, Aguilar-López, R, Cebrián-García, ME
Preparative biochemistry & biotechnology. 2022;(3):292-301
Abstract
This study evaluates the production of a biological active surface agent (BASA) through its surface tension (ST) and emulsifying activity (E24) for endosulfan degradation (ED) and Escherichia coli growth inhibition (EcGI) in an agricultural saline soil. The fungus, identified as Penicillium crustosum was isolated from the Citrus sinensis peel (CsP), then the surface properties were evaluated in 9 culture media through a Taguchi L9 experimental design. The culture conditions included: stirring speed, pH, carbon (C) and nitrogen (N) sources; being glucose, NH4N03, 120 rpm and pH of 5, the most significant parameters in the BASA production. The BASA identified as a lipopeptide type, showed a ST = 38 mN m-1 and E24=71%. Both properties were stable at 80 °C, while ST presented stability in the pH range of 2 - 12, and a saline concentration of 200 g L-1; E24 was also stable at a pH between 8-12. Further application of BASA and fungal inoculum to a contaminated agricultural saline soil presented an EcGI of 99.8% on the 8th day, and ED of 92.9 ± 4.7% in 30 days, respectively; being the first report that uses this fungus for pesticide and bacteria elimination from an agricultural saline soil.
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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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High-mechanical strength carboxymethyl chitosan-based hydrogel film for antibacterial wound dressing.
Zhang, M, Yang, M, Woo, MW, Li, Y, Han, W, Dang, X
Carbohydrate polymers. 2021;:117590
Abstract
Hydrogels, being highly biocompatible and adaptable with biological tissues, have shown great usability in biomedical applications. In this research, a novel hydrogel film developed from carboxymethyl chitosan (CMCS) loaded with waterborne polyurethane-gelatin hydrolysate was synthesized via aqueous emulsion copolymerization. The synthesized hydrogel film was characterized using mechanical strength tests, FTIR, XPS, SEM, AFM, and various other analysis technologies. The results demonstrated that the hydrogel film exhibited good thermal stability, swelling behavior, as well as controllable biodegradability. Specifically, when the CMCS content was loaded at 6 %, the maximum tensile strength and elongation at the break of the hydrogel film were reached 31.69 MPa and 447.187, respectively. The disk diffusion tests indicated that the hydrogel film presented significant antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). These results indicate that hydrogel films with high mechanical strength and high antibacterial activity could be used for wound dressing applications.
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Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.
Kutyshenko, VP, Prokhorov, DA, Mikoulinskaia, GV, Molochkov, NV, Yegorov, AY, Paskevich, SI, Uversky, VN
International journal of biological macromolecules. 2021;:1096-1105
Abstract
The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.
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7.
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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Abstract
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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8.
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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9.
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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10.
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.