RESUMO
The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun's lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.
Assuntos
Proteínas de Escherichia coli , Peptidoglicano , Antibacterianos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte , Parede Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Peptidoglicano/metabolismoRESUMO
Predicting the range of substrates accepted by an enzyme from its amino acid sequence is challenging. Although sequence- and structure-based annotation approaches are often accurate for predicting broad categories of substrate specificity, they generally cannot predict which specific molecules will be accepted as substrates for a given enzyme, particularly within a class of closely related molecules. Combining targeted experimental activity data with structural modeling, ligand docking, and physicochemical properties of proteins and ligands with various machine learning models provides complementary information that can lead to accurate predictions of substrate scope for related enzymes. Here we describe such an approach that can predict the substrate scope of bacterial nitrilases, which catalyze the hydrolysis of nitrile compounds to the corresponding carboxylic acids and ammonia. Each of the four machine learning models (logistic regression, random forest, gradient-boosted decision trees, and support vector machines) performed similarly (average ROC = 0.9, average accuracy = ~82%) for predicting substrate scope for this dataset, although random forest offers some advantages. This approach is intended to be highly modular with respect to physicochemical property calculations and software used for structural modeling and docking.
Assuntos
Aminoidrolases , Proteínas de Bactérias , Aprendizado de Máquina , Simulação de Acoplamento Molecular/métodos , Aminoidrolases/química , Aminoidrolases/genética , Aminoidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Fenômenos Químicos , Ligantes , Nitrilas/química , Nitrilas/metabolismo , Ligação ProteicaRESUMO
Mercury (Hg) pollution is a global environmental problem. The abiotic formation of dimethylmercury (DMeHg) from monomethylmercury (MMeHg) may account for a large portion of DMeHg in oceans. Previous experimental work has shown that abiotic formation of DMeHg from MMeHg can be facilitated by reduced sulfur groups on sulfide mineral surfaces. In that work, a mechanism was proposed in which neighboring MMeHg moieties bound to sulfide sites on a mineral surface react through an SN2-type mechanism to form DMeHg and incorporate the remaining Hg atoms into the mineral surface. Here, we perform density functional theory calculations to explore the mechanisms of DMeHg formation on the 110 surface of a CdS(s) (hawleyite) nanoparticle. We show that coordination of MMeHg substituents to adjacent reduced sulfur groups protruding from the surface indeed facilitates DMeHg formation and that the reaction proceeds through direct transmethylation from one MMeHg substituent to another. Coordination of Hg by multiple S atoms provides a transition-state stabilization and activates a C-Hg bond for methyl transfer. In addition, solvation effects play an important role in the surface reconstruction of the nanoparticle and in decreasing the energetic barrier for DMeHg formation relative to the corresponding reaction in vacuo.
RESUMO
A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.
Assuntos
Ácidos Cumáricos/metabolismo , Nucleotídeos de Purina/biossíntese , Acinetobacter baumannii/metabolismo , Escherichia coli/genética , Evolução Molecular , Técnicas de Transferência de Genes , Transferência Genética Horizontal , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Redes e Vias Metabólicas/genética , Simulação de Dinâmica Molecular , Mutação , Nucleotídeos de Purina/antagonistas & inibidores , Nucleotídeos de Purina/genéticaRESUMO
Mercury (Hg) is a global environmental contaminant. Major anthropogenic sources of Hg emission include gold mining and the burning of fossil fuels. Once deposited in aquatic environments, Hg can undergo redox reactions, form complexes with ligands, and adsorb onto particles. It can also be methylated by microorganisms. Mercury, especially its methylated form methylmercury, can be taken up by organisms, where it bioaccumulates and biomagnifies in the food chain, leading to detrimental effects on ecosystem and human health. In support of the recently enforced Minamata Convention on Mercury, a legally binding international convention aimed at reducing the anthropogenic emission of-and human exposure to-Hg, its global biogeochemical cycle must be understood. Thus, a detailed understanding of the molecular-level interactions of Hg is crucial. The ongoing rapid development of hardware and methods has brought computational chemistry to a point that it can usefully inform environmental science. This is particularly true for Hg, which is difficult to handle experimentally due to its ultratrace concentrations in the environment and its toxicity. The current account provides a synopsis of the application of computational chemistry to filling several major knowledge gaps in environmental Hg chemistry that have not been adequately addressed experimentally. Environmental Hg chemistry requires defining the factors that determine the relative affinities of different ligands for Hg species, as they are critical for understanding its speciation, transformation and bioaccumulation in the environment. Formation constants and the nature of bonding have been determined computationally for environmentally relevant Hg(II) complexes such as chlorides, hydroxides, sulfides and selenides, in various physical phases. Quantum chemistry has been used to determine the driving forces behind the speciation of Hg with hydrochalcogenide and halide ligands. Of particular importance is the detailed characterization of solvation effects. Indeed, the aqueous phase reverses trends in affinities found computationally in the gas phase. Computation has also been used to investigate complexes of methylmercury with (seleno)amino acids, providing a molecular-level understanding of the toxicological antagonism between Hg and selenium (Se). Furthermore, evidence is emerging that ice surfaces play an important role in Hg transport and transformation in polar and alpine regions. Therefore, the diffusion of Hg and its ions through an idealized ice surface has been characterized. Microorganisms are major players in environmental mercury cycling. Some methylate inorganic Hg species, whereas others demethylate methylmercury. Quantum chemistry has been used to investigate catalytic mechanisms of enzymatic Hg methylation and demethylation. The complex interplay between the myriad chemical reactions and transport properties both in and outside microbial cells determines net biogeochemical cycling. Prospects for scaling up molecular work to obtain a mechanistic understanding of Hg cycling with comprehensive multiscale biogeochemical modeling are also discussed.
Assuntos
Poluentes Ambientais/química , Mercúrio/química , Química Computacional/métodos , Simulação por Computador , Difusão , Metilação , Metiltransferases/química , Modelos Moleculares , Oxirredutases/química , Termodinâmica , Água/químicaRESUMO
Cellular uptake and export are important steps in the biotransformation of mercury (Hg) by microorganisms. However, the mechanisms of transport across biological membranes remain unclear. Membrane-bound transporters are known to be relevant, but passive permeation may also be involved. Inorganic HgII and methylmercury ([CH3HgII]+) are commonly complexed with thiolate ligands. Here, we have performed extensive molecular dynamics simulations of the passive permeation of HgII and [CH3HgII]+ complexes with thiolate ligands through a model bacterial cytoplasmic membrane. We find that the differences in free energy between the individual complexes in bulk water and at their most favorable position within the membrane are â¼2 kcal mol-1. We provide a detailed description of the molecular interactions that drive the membrane crossing process. Favorable interactions with carbonyl and tail groups of phospholipids stabilize Hg-containing solutes in the tail-head interface region of the membrane. The calculated permeability coefficients for the neutral compounds CH3S-HgII-SCH3 and CH3HgII-SCH3 are on the order of 10-5 cm s-1. We conclude that small, nonionized Hg-containing species can permeate readily through cytoplasmic membranes.
Assuntos
Membrana Celular , Mercúrio/farmacocinética , Compostos de Metilmercúrio/farmacocinética , Modelos Biológicos , Fosfolipídeos , ÁguaRESUMO
Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.
Assuntos
COVID-19 , Hepatite C Crônica , Animais , Humanos , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases , Mamíferos/metabolismoRESUMO
Optimization of compound permeation into Gram-negative bacteria is one of the most challenging tasks in the development of antibacterial agents. Two permeability barriersâthe passive diffusion barrier of the outer membrane (OM) and active drug effluxâact synergistically to protect cells from the antibacterial action of compounds. In Escherichia coli (E. coli) and relatives, these two barriers sieve compounds based on different physicochemical properties that are defined by their interactions with OM porins and efflux pumps, respectively. In this study, we critically tested the hypothesis that the best substrates and inhibitors of efflux pumps are compounds that can effectively permeate the OM and are available at relatively high concentrations in the periplasm. For this purpose, we filtered a large subset of the ZINC15 database of commercially available compounds for compounds containing a primary amine, a chemical feature known to facilitate the uptake through E. coli general porins. The assembled library was screened by ensemble docking to AcrA, the periplasmic component of the AcrAB-TolC efflux pump, followed by experimental testing of the top predicted binders for antibacterial activities, efflux recognition, and inhibition. We found that the filtered primary amine library is a rich source of compounds with efflux-inhibiting activities and identified efflux pump inhibitors with novel chemical scaffolds effective against E. coli AcrAB-TolC and efflux pumps of multidrug-resistant clinical isolates of Acinetobacter baumannii. However, primary amines are not required for the recognition of compounds by efflux pumps and their efflux-inhibitory activities.
Assuntos
Escherichia coli , Proteínas de Membrana Transportadoras , Aminas , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Membrana Transportadoras/química , PorinasRESUMO
Photosystem I (PSI) is one of two photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutionary significance. Here we report the â¼3.72 Å resolution cryo-electron microscopy structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in the PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.
Assuntos
Cianobactérias , Complexo de Proteína do Fotossistema I , Clorofila , Microscopia Crioeletrônica , Cianobactérias/química , Cianobactérias/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismoRESUMO
Two membrane cell envelopes act as selective permeability barriers in Gram-negative bacteria, protecting cells against antibiotics and other small molecules. Significant efforts are being directed toward understanding how small molecules permeate these barriers. In this study, we developed an approach to analyze the permeation of compounds into Gram-negative bacteria and applied it to Pseudomonas aeruginosa, an important human pathogen notorious for resistance to multiple antibiotics. The approach uses mass spectrometric measurements of accumulation of a library of structurally diverse compounds in four isogenic strains of P. aeruginosa with varied permeability barriers. We further developed a machine learning algorithm that generates a deterministic classification model with minimal synonymity between the descriptors. This model predicted good permeators into P. aeruginosa with an accuracy of 89% and precision above 58%. The good permeators are broadly distributed in the property space and can be mapped to six distinct regions representing diverse chemical scaffolds. We posit that this approach can be used for more detailed mapping of the property space and for rational design of compounds with high Gram-negative permeability.
Assuntos
Bactérias Gram-Negativas , Pseudomonas aeruginosa , Antibacterianos/química , Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Permeabilidade , Pseudomonas aeruginosa/metabolismoRESUMO
Protein-protein interactions play a key role in mediating numerous biological functions, with more than half the proteins in living organisms existing as either homo- or hetero-oligomeric assemblies. Protein subunits that form oligomers minimize the free energy of the complex, but exhaustive computational search-based docking methods have not comprehensively addressed the challenge of distinguishing a natively bound complex from non-native forms. Current protein docking approaches address this problem by sampling multiple binding modes in proteins and scoring each mode, with the lowest-energy (or highest scoring) binding mode being regarded as a near-native complex. However, high-scoring modes often match poorly with the true bound form, suggesting a need for improvement of the scoring function. In this study, we propose a scoring function, KFC-E, that accounts for both conservation and coevolution of putative binding hotspot residues at protein-protein interfaces. We tested KFC-E on four benchmark sets of unbound examples and two benchmark sets of bound examples, with the results demonstrating a clear improvement over scores that examine conservation and coevolution across the entire interface.
Assuntos
Proteínas , Ligação Proteica , Conformação Proteica , Proteínas/metabolismoRESUMO
Antibiotic resistance poses an immediate and growing threat to human health. Multidrug efflux pumps are promising targets for overcoming antibiotic resistance with small-molecule therapeutics. Previously, we identified a diaminoquinoline acrylamide, NSC-33353, as a potent inhibitor of the AcrAB-TolC efflux pump in Escherichia coli. This inhibitor potentiates the antibacterial activities of novobiocin and erythromycin upon binding to the membrane fusion protein AcrA. It is also a substrate for efflux and lacks appreciable intrinsic antibacterial activity of its own in wild-type cells. Here, we have modified the substituents of the cinnamoyl group of NSC-33353, giving rise to analogs that retain the ability to inhibit efflux, lost the features of the efflux substrates, and gained antibacterial activity in wild-type cells. The replacement of the cinnamoyl group with naphthyl isosteres generated compounds that lack antibacterial activity but are both excellent efflux pump inhibitors and substrates. Surprisingly, these inhibitors potentiate the antibacterial activity of novobiocin but not erythromycin. Surface plasmon resonance experiments and molecular docking suggest that the replacement of the cinnamoyl group with naphthyl shifts the affinity of the compounds away from AcrA to the AcrB transporter, making them better efflux substrates and changing their mechanism of inhibition. These results provide new insights into the duality of efflux substrate/inhibitor features in chemical scaffolds that will facilitate the development of new efflux pump inhibitors.
Assuntos
Proteínas de Escherichia coli , Amidas/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that ß-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the ß-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.
Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Coxiella burnetii/metabolismo , Escherichia coli/metabolismo , Legionella pneumophila/metabolismo , Peptidoglicano/metabolismo , Ciclo Celular/fisiologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Lipoproteínas/metabolismo , Simulação de Dinâmica Molecular , Peptidil Transferases/metabolismo , Ligação Proteica/fisiologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T-cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune resistance in pancreatic cancer was induced in response to antitumor T-cell immune responses and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide evidence that T-cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune resistance. Three KPC mouse models of pancreatic cancer were used: the mT3-2D (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) subcutaneous and orthotopic models, as well as the KP1 (p48-CRE/LSL-Kras/Trp53 flox/flox ) subcutaneous model. KPC cancer cells were grown in immunocompetent and immunodeficient C57BL/6 mice and analyzed to determine the impact of adaptive immunity on malignant epithelial cells, as well as on whole tumors. We found that induced T-cell antitumor immunity, via signal transducer and activator of transcription 1 (STAT1), stimulated malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and altered intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA-approved drug ruxolitinib overcame these tumor-protective responses and improved anti-PD-1 therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Modelos Animais de Doenças , Nitrilas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Linfócitos T/imunologia , Animais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral/transplante , Humanos , Metalotioneína 3 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Pâncreas/imunologia , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Microambiente Tumoral , Ubiquitina-Proteína LigasesRESUMO
Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. Here we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Lipoproteínas/química , Proteínas de Membrana Transportadoras/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/patogenicidade , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Anti-Infecciosos/química , Proteínas de Transporte/antagonistas & inibidores , Biologia Computacional/métodos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Sinergismo Farmacológico , Eritromicina/química , Eritromicina/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/patologia , Humanos , Klebsiella pneumoniae , Lipoproteínas/antagonistas & inibidores , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Novobiocina/química , Novobiocina/farmacologiaRESUMO
Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mercúrio/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Bactérias/genética , Corrinoides/metabolismo , Desulfovibrio desulfuricans/genética , Metagenoma , Metilação , Modelos Moleculares , Complexos Multiproteicos/genética , Filogenia , Conformação Proteica , Domínios Proteicos , Espectrofotometria UltravioletaRESUMO
Bio-based poly(butylene succinate) (BioPBS) was combined with pyrolyzed Miscanthus microparticles (biocarbon) and graphene nanoplatelets to create a hybrid bionanocomposite. Pyrolyzed biomass, known as biocarbon, was incorporated into a BioPBS matrix to improve the thermo-mechanical properties of the bioplastic while simultaneously increasing the value of this co-product. Biocomposites loaded with 25 wt % biocarbon showed 57, 13, and 32% improvements in tensile modulus, heat deflection temperature, and thermal expansion, respectively. Further improvements were found when graphene nanoplatelets (GnPs) were added to the biocomposite, forming a hierarchical hybrid bionanocomposite. Two processing methods were used to incorporate graphene into the composites: (I) graphene, BioPBS, and biocarbon were added together and directly compounded, and (II) a masterbatch of graphene and BioPBS was processed first and then diluted to the same ratios as those used in the direct compounding method I. The two methods resulted in different internal morphologies that subsequently impacted the mechanical properties of the composites; little change was observed in the thermal properties studied. Bionanocomposites processed using the direct compounding technique showed the greatest increase in tensile strength and modulus: 17 and 120%, respectively. Bionanocomposites processed using a masterbatch technique had slightly lower strength and modulus but showed almost twice the impact strength compared with the direct compounding method. This masterbatch technique was found to have a superior balance of stiffness and toughness, likely due to the presence of superclustered graphene platelets, confirmed through a scanning electron microscope and a transmission electron microscope.
RESUMO
Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.
Assuntos
Actinobacteria/metabolismo , Anticorpos/metabolismo , Proteínas de Membrana/imunologia , Boca/microbiologia , Análise de Célula Única/métodos , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Genômica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Filogenia , Conformação Proteica , Genética Reversa , Análise de Sequência de DNARESUMO
Biobased poly(butylene succinate) (BioPBS) was electrospun to create hierarchical, highly porous fibers. Various grades of BioPBS were dissolved in one of the three solutions: chloroform, a co-solvent system of chloroform/N,N-dimethylformamide (DMF), or chloroform/dimethyl sulfoxide (DMSO). These solutions were then electrospun at room temperature to produce nanoporous micron-sized fibers. The variables investigated were the solvent system used, grade of BioPBS, concentration of BioPBS, applied voltage, and the distance between the electrodes. In determining the optimal solution and electrospinning conditions, it was found that solution properties such as the solvent system, the grade of BioPBS, and the concentration of BioPBS had a significant effect on the fiber morphology. A chloroform/DMSO cosolvent system resulted in less bead defects among fibers compared to those produced from chloroform/DMF systems, regardless of the BioPBS grade. An increase in BioPBS concentration resulted in the reduction of bead defects, which at 15 (% w/v) resulted in bead-free uniform fibers. Increasing BioPBS concentration also increased the porosity of the fibers while reducing the pore size. Dynamic mechanical analysis showed that the reduction of bead defects resulted in increased tensile strength and Young's modulus of the electrospun fibrous nonwoven mat. The results of this study show that electrospun BioPBS fibers have high porosity at the micro- and nanoscale, resulting in a hierarchical structure that has sufficient mechanical properties for potential applications in wound healing and soft tissue engineering.
RESUMO
The permeability barrier of Gram-negative cell envelopes is the major obstacle in the discovery and development of new antibiotics. In Gram-negative bacteria, these difficulties are exacerbated by the synergistic interaction between two biochemically distinct phenomena, the low permeability of the outer membrane (OM) and active multidrug efflux. In this study, we used Pseudomonas aeruginosa and Escherichia coli strains with controllable permeability barriers, achieved through hyperporination of the OMs and varied efflux capacities, to evaluate the contributions of each of the barriers to protection from antibacterials. We analyzed antibacterial activities of ß-lactams and fluoroquinolones, antibiotics that are optimized for targets in the periplasm and the cytoplasm, respectively, and performed a machine learning-based analysis to identify physicochemical descriptors that best classify their relative potencies. Our results show that the molecular properties selected by active efflux and the OM barriers are different for the two species. Antibiotic activity in P. aeruginosa was better classified by electrostatic and surface area properties, whereas topology, physical properties, and atom or bond counts best capture the behavior in E. coli. In several cases, descriptor values that correspond to active antibiotics also correspond to significant barrier effects, highlighting the synergy between the two barriers where optimizing for one barrier promotes strengthening of the other barrier. Thus, both barriers should be considered when optimizing antibiotics for favorable OM permeability, efflux evasion, or both.