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In biogeochemical phosphorus cycling, iron oxide minerals are acknowledged as strong adsorbents of inorganic and organic phosphorus. Dephosphorylation of organic phosphorus is attributed only to biological processes, but iron oxides could also catalyze this reaction. Evidence of this abiotic catalysis has relied on monitoring products in solution, thereby ignoring iron oxides as both catalysts and adsorbents. Here we apply high-resolution mass spectrometry and X-ray absorption spectroscopy to characterize dissolved and particulate phosphorus species, respectively. In soil and sediment samples reacted with ribonucleotides, we uncover the abiotic production of particulate inorganic phosphate associated specifically with iron oxides. Reactions of various organic phosphorus compounds with the different minerals identified in the environmental samples reveal up to ten-fold greater catalytic reactivities with iron oxides than with silicate and aluminosilicate minerals. Importantly, accounting for inorganic phosphate both in solution and mineral-bound, the dephosphorylarion rates of iron oxides were within reported enzymatic rates in soils. Our findings thus imply a missing abiotic axiom for organic phosphorus mineralization in phosphorus cycling.
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Sugarcane bagasse and rice straw are major agricultural byproducts often discarded or burned as waste after cultivation, leaving their untapped potential for utilization. In this work, cellulose fibers were extracted from sugarcane bagasse and rice straw using a simple procedure: alkaline treatment with sodium hydroxide, bleaching with sodium hypochlorite, and acid hydrolysis. The obtained cellulosic materials were successfully prepared into milky white and transparent films, of which the transparency slightly decreased with the addition of glycerol. The surface of all the films appeared homogeneous with a random orientation of fibers. The rice-straw (RS) film had a more fragile texture than the sugarcane-bagasse (SBG) film. The FTIR analysis clearly indicated the functional groups of cellulose, as well as glycerol for the films mixed with glycerol. Thermal analysis showed that the native SBG film decomposed at 346 °C, higher than the native RS film (339 °C). The presence of glycerol in the films resulted in slightly lower maximum decomposition temperature (Td,max) values as well as mechanical properties. Regarding water susceptibility, the RS film had a higher percentage than the native SBG and glycerol-mixed SBG films. The extracted cellulose from both sources could form almost spherical-shaped cellulose particles. Thus, through the simple extraction method, sugarcane bagasse and rice straw could serve as excellent sources of cellulose materials for preparing cellulose films and particles, which would be advantageous to the development of cellulose-based materials.
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Poly(L-lactide) (PLLA) is a promising candidate as a bioplastic because of its non-toxicity and biodegradability. However, the low flexibility of PLLA limits its use in many applications. Poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-b-PEG-b-PLLA) block copolymer is of interest for bioplastic applications due to its superior flexibility compared to PLLA. The aim of this work is to modify PLLA-b-PEG-b-PLLA using a low-cost calcium carbonate (CaCO3) filler to improve material properties compared to PLLA/CaCO3 composites. The addition of CaCO3 enhanced the crystallinity and thermal stability for the PLLA-b-PEG-b-PLLA matrix but not for the PLLA matrix, as determined by differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and thermogravimetric analysis (TGA). Phase morphology investigation using scanning electron microscopy (SEM) revealed that the interfacial adhesion between PLLA-b-PEG-b-PLLA and CaCO3 was stronger than between PLLA and CaCO3. Additionally, tensile testing was carried out to determine the mechanical properties of the composites. With the addition of CaCO3, the tensile stress and Young's modulus of the PLLA-b-PEG-b-PLLA matrix were increased, whereas these properties of the PLLA matrix were significantly decreased. Thus, CaCO3 shows great promise as an inexpensive filler that can induce nucleation and reinforcing effects for PLLA-b-PEG-b-PLLA bioplastics.
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Poly(l-lactide)-b-poly(ethylene glycol)-b-poly(l-lactide) block copolymer (PLLA-PEG-PLLA) is a highly flexible bioplastic, yet its use in practical applications is limited due to its poor heat resistance and high production cost. In this study, talcum was used as a nucleating agent to improve the heat resistance, and thermoplastic starch (TPS) was used as a low-cost filler to reduce the cost of production. PLLA-PEG-PLLA/talcum/TPS and PLLA/talcum/TPS ternary composites with 4 wt% talcum and various TPS contents were prepared by melt blending before injection molding and were then evaluated. When PEG middle-blocks were present, the PLLA-PEG-PLLA-based composites showed a higher crystallinity, more flexibility, and a higher heat resistance than the PLLA-based composites. Although the addition of TPS decreased the heat resistance of all the composites, the PLLA-PEG-PLLA/talcum/TPS composites still had high Vicat softening temperatures (VST, 113-131 °C) and demonstrated a good dimensional stability to heat by maintaining their original shapes upon heat exposure. The biodegradation test in soil suggested that the synergistic effect of the PEG middle-blocks and TPS significantly increased the biodegradability of the PLLA-PEG-PLLA/talcum/TPS composites. This improved heat resistance, lower cost, and accelerated biodegradation make PLLA-PEG-PLLA/talcum/TPS composites a promising material to be used as heat-resistant and single-use bioplastic products.
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Temperatura Alta , Amido , Polietilenoglicóis , PoliésteresRESUMO
Critical to meeting cellular phosphorus (P) demand, soil bacteria deploy a number of strategies to overcome limitation in inorganic P (Pi ) in soils. As a significant contributor to P recycling, soil bacteria secrete extracellular enzymes to degrade organic P (Po ) in soils into the readily bioavailable Pi . In addition, several Po compounds can be transported directly via specific transporters and subsequently enter intracellular metabolic pathways. In this review, we highlight the strategies that soil bacteria employ to recycle Po from the soil environment. We discuss the diversity of extracellular phosphatases in soils, the selectivity of these enzymes towards various Po biomolecules and the influence of the soil environmental conditions on the enzyme's activities. Moreover, we outline the intracellular metabolic pathways for Po biosynthesis and transporter-assisted Po and Pi uptake at different Pi availabilities. We further highlight the regulatory mechanisms that govern the production of phosphatases, the expression of Po transporters and the key metabolic changes in P metabolism in response to environmental Pi availability. Due to the depletion of natural resources for Pi , we propose future studies needed to leverage bacteria-mediated P recycling from the large pools of Po in soils or organic wastes to benefit agricultural productivity.
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Fósforo , Solo , Agricultura , Bactérias/genética , Bactérias/metabolismo , Fósforo/metabolismo , Microbiologia do SoloRESUMO
Bacterial biofilms are communities of bacteria entangled in a self-produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid-polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum-of-the-parts 13 C solid-state nuclear magnetic resonance (NMR) analysis to define the curli-to-pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well-studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid-air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic-associated) and the strain itself.
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Proteínas de Bactérias/química , Biofilmes , Celulose/química , Matriz Extracelular/química , Biomassa , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Etanolaminas/químicaRESUMO
Bacterial biofilms are surface-associated communities of bacterial cells enmeshed in an extracellular matrix (ECM). The biofilm lifestyle results in physiological heterogeneity across the community, promotes persistence, and protects cells from external insults such as antibiotic treatment. Escherichia coli was recently discovered to produce a chemically modified form of cellulose, phosphoethanolamine (pEtN) cellulose, which contributes to the formation of its extracellular matrix and elaboration of its hallmark wrinkled macrocolony architectures. Both pEtN cellulose and unmodified cellulose bind dyes such as calcofluor white and Congo red (CR). Here, we present the use of CR fluorescence to distinguish between pEtN cellulose and unmodified cellulose producers. We demonstrate the utility of this tool in the evaluation of a uropathogenic E. coli clinical isolate that appeared to produce curli and a cellulosic component but did not exhibit macrocolony wrinkling. We determined that lack of macrocolony wrinkling was attributed to a single-nucleotide mutation and introduction of a stop codon in bcsG, abrogating production of BcsG, the pEtN transferase. Thus, this work underscores the important contribution of the pEtN cellulose modification to the E. coli agar-based macrocolony wrinkling phenotype and introduces a facile approach to distinguish between modified and unmodified cellulose.IMPORTANCEE. coli bacteria produce amyloid fibers, termed curli, and a cellulosic component to assemble biofilm communities. Cellulose is the most abundant biopolymer on Earth, and we recently discovered that the cellulosic component in E. coli biofilms was not standard cellulose, but a newly identified cellulosic polymer, phosphoethanolamine cellulose. Studies involving the biological and functional impact of this cellulose modification among E. coli and other organisms are just beginning. Convenient methods for distinguishing pEtN cellulose from unmodified cellulose in E. coli and for estimating production are needed to facilitate further research. Dissecting the balance of pEtN cellulose and curli production by E. coli commensal strains and clinical isolates will improve our understanding of the host microbiome and of factors contributing to bacterial pathogenesis.
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Celulose/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Coloração e Rotulagem/métodos , Celulose/química , Vermelho Congo/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Etanolaminas/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , FluorescênciaRESUMO
Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.
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Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Vibrio cholerae/enzimologia , Vibrio cholerae/fisiologia , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Locomoção , Fósforo-Oxigênio Liases/genética , Vibrio cholerae/genéticaRESUMO
Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.
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Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Celulose/efeitos adversos , Células Epiteliais/metabolismo , Etanolaminas/efeitos adversos , Bexiga Urinária/metabolismo , Escherichia coli Uropatogênica/metabolismo , Urotélio/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Celulose/farmacologia , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Etanolaminas/farmacologia , Humanos , Bexiga Urinária/microbiologia , Bexiga Urinária/ultraestrutura , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/ultraestrutura , Urotélio/microbiologia , Urotélio/ultraestruturaRESUMO
Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.
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Celulose/biossíntese , Celulose/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Etanolaminas/metabolismo , Óperon/fisiologia , Biofilmes , Celulose/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Etanolaminas/química , Óperon/genéticaRESUMO
Microbial biofilms are communities of cells characterized by a hallmark extracellular matrix (ECM) that confers functional attributes to the community, including enhanced cohesion, adherence to surfaces, and resistance to external stresses. Understanding the composition and properties of the biofilm ECM is crucial to understanding how it functions and protects cells. New methods to isolate and characterize ECM are emerging for different biofilm systems. Solid-state nuclear magnetic resonance was used to quantitatively track the isolation of the insoluble ECM from the uropathogenic Escherichia coli strain UTI89 and understand the role of Congo red in purification protocols. UTI89 assembles amyloid-integrated biofilms when grown on YESCA nutrient agar. The ECM contains curli amyloid fibers and a modified form of cellulose. Biofilms formed by UTI89 and other E. coli and Salmonella strains are often grown in the presence of Congo red to visually emphasize wrinkled agar morphologies and to score the production of ECM. Congo red is a hallmark amyloid-binding dye and binds to curli, yet also binds to cellulose. We found that growth in Congo red enabled more facile extraction of the ECM from UTI89 biofilms and facilitates isolation of cellulose from the curli mutant, UTI89ΔcsgA. Yet, Congo red has no influence on the isolation of curli from curli-producing cells that do not produce cellulose. Sodium dodecyl sulfate can remove Congo red from curli, but not from cellulose. Thus, Congo red binds strongly to cellulose and possibly weakens cellulose interactions with the cell surface, enabling more complete removal of the ECM. The use of Congo red as an extracellular matrix purification aid may be applied broadly to other organisms that assemble extracellular amyloid or cellulosic materials. Graphical abstract Solid-state NMR was used to quantitatively track the isolation of the insoluble amyloid-associated ECM from uropathogenic E. coli and understand the role of Congo red in purification protocols.
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Amiloide/química , Biofilmes/efeitos dos fármacos , Vermelho Congo/química , Matriz Extracelular/química , Escherichia coli Uropatogênica/efeitos dos fármacos , Amiloide/biossíntese , Amiloide/ultraestrutura , Biofilmes/crescimento & desenvolvimento , Celulose/química , Vermelho Congo/farmacologia , Meios de Cultura/química , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/ultraestruturaRESUMO
In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.
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GMP Cíclico/análogos & derivados , Proteínas de Fímbrias/biossíntese , Fímbrias Bacterianas/efeitos dos fármacos , Vibrio cholerae/efeitos dos fármacos , Biofilmes , GMP Cíclico/farmacologia , Epistasia Genética , Fímbrias Bacterianas/fisiologia , Lectina de Ligação a Manose/biossíntese , Movimento , Vibrio cholerae/fisiologiaRESUMO
The introduction of reversible covalent bonds into polymeric systems afford robust, yet dynamic, materials that can respond to external stimuli. A series of aliphatic polycarbonate polymers were synthesized via ring-opening polymerization of furanyl and maleimido-bearing cyclic carbonate monomers. These side chains undergo thermally induced Diels-Alder reactions to afford cross-linked films. Because both the diene and dienophile were incorporated into the same polymer backbone, a protected maleimido group, in the form of the furan adduct, was used. Both the forward and reverse Diels-Alder reaction are triggered thermally, which allows the deprotection of the maleimido group and the subsequent reaction with the furanyl side chains to form cross-links. Random copolymers and poly(ethylene glycol) containing block copolymers were formed using diazabicyclo[5.4.0]undec-7-ene as the catalyst and a thiourea cocatalyst. The polymers form uniform films that can be cross-linked in the bulk state. To further illustrate the dynamic nature of the covalent bonds within the cross-linked films, a patterned silicon mold was used to transfer a series of nanoscale patterns using a thermal nanoimprint process.