RESUMEN
Biopolymers on the cell surface are very important for protecting microorganisms from environmental stresses, as well as storing nutrients and minerals. Synthesis of biopolymers is well studied, while studies on the modification and degradation processes of biopolymers are limited. One of these biopolymers, poly-γ-glutamic acid (γ-PGA), is produced by Bacillus species. Bacillus subtilis PgdS, possessing three NlpC/P60 domains, hydrolyses γ-PGA. Here, we have demonstrated that several dl-endopeptidases with an NlpC/P60 domain (LytE, LytF, CwlS, CwlO, and CwlT) in B. subtilis digest not only an amide bond of d-γ-glutamyl-diaminopimelic acid in peptidoglycans but also linkages of γ-PGA produced by B. subtilis. The hydrolase activity of dl-endopeptidases towards γ-PGA was inhibited by IseA, which also inhibits their hydrolase activity towards peptidoglycans, while the hydrolysis of PgdS towards γ-PGA was not inhibited. PgdS hydrolysed only the d-/l-Gluâd-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7â:â3) and l-Glu-rich γ-PGA (d-Glu:l-Glu=1â:â9), indicating that PgdS can hydrolyse only restricted substrates. On the other hand, the dl-endopeptidases in B. subtilis cleaved d-/l-Gluâd-/l-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7â:â3), indicating that these enzymes show different substrate specificities. Thus, the dl-endopeptidases digest γ-PGA more flexibly than PgdS, even though they are annotated as "dl-endopeptidase, digesting the d-γ-glutamyl-diaminopimelic acid linkage (dâl amino acid bond)".
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Pared Celular/enzimología , Endopeptidasas/metabolismo , Hidrolasas/metabolismo , Ácido Poliglutámico/análogos & derivados , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Biopolímeros/metabolismo , Dominio Catalítico , Pared Celular/metabolismo , Endopeptidasas/química , Hidrólisis , Peptidoglicano/metabolismo , Ácido Poliglutámico/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
The low volatility of ionic liquids (ILs) is one of their most interesting physico-chemical properties; however, the general understanding of their evaporation dynamics under vacuum is still lagging. Here, we studied the thermodynamics of IL evaporation by employing thermogravimetry (TG) measurements under vacuum. The thermodynamic parameters of ILs, such as the evaporation onset temperatures, enthalpies, entropies, saturation vapor pressures, and boiling points were quantified by analyzing the TG data. The obtained evaporation enthalpies (110-140 kJ mol-1) were higher than those of typical molecular liquids, and the entropies (>88 J mol-1 K-1) suggested that they are exceptions of the Trouton's rule. The obtained Clausius-Clapeyron equations demonstrated that the saturation vapor pressures of ILs only depend on temperature. Further, we derived the empirical equation for estimating the upper limit temperature of the liquid phase of IL under given external pressures. Using the evaporation behaviors of referential normal alkanes and charge-transfer complex and the evaporation entropies of the ILs, the vaporized IL structure was thermodynamically modelled. The ILs were found to evaporate as ion pairs, instead of as individual ions or higher-ordered cluster structures. By comparing a series of ILs with various cations and a fixed anion, it was found that the IL evaporation dynamics under vacuum is strongly and systematically affected by their chemical structures, charge balances between the cations and the anions, molecular weights, and the higher-ordered structures including polar and non-polar regions. Our concept, measurement method, and equation can be extended to other ILs and low-volatile liquids under vacuum, and help with the design of ILs with higher thermal stabilities.
RESUMEN
[10]Cycloparaphenylene ([10]CPP) and its tetraalkoxy derivatives were synthesized on the gram scale in 7 steps starting from 1,4-benzoquinone or 2,5-dialkoxy-1,4-benzoquinone. The key steps involve the highly cis-selective bis-addition of 4-bromo-4'-lithiobiphenyl to the quinones to produce a five-ring unit containing cyclohexa-1,4-diene-3,6-diol moiety, the platinum-mediated dimerization of the five-ring unit, and the H2SnCl4-mediated reductive aromatization of cyclohexadienediol. The tetraalkoxy substituents increased the solubility of [10]CPP in common organic solvents. The carrier-transport properties of thin films of [10]CPP and its derivatives were measured for the first time and indicated that [10]CPP derivatives could rival phenyl-C61-butyric acid methyl ester, which is used widely as an n-type active layer in bulk heterojunction photovoltaics.
RESUMEN
Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic-biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe2O3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe2O3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe2O3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF-nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe2O3 follows a strategy to connect proteins to materials that resembles the binding between donor-acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein-nanoparticle binding at the molecular level, this work reveals one of nature's strategies for achieving fast, efficient electron transfer between proteins and materials.
RESUMEN
Molecular orientation in amorphous organic semiconducting thin-film devices is an important issue affecting device performance. However, to date it has not been possible to analyze the "distribution" of the orientations. Although solid-state NMR (ssNMR) spectroscopy can provide information on the "distribution" of molecular orientations, the technique is limited because of the small amount of sample in the device and the low sensitivity of ssNMR. Here, we report the first application of dynamic nuclear polarization enhanced ssNMR (DNP-ssNMR) spectroscopy for the orientational analysis of amorphous phenyldi(pyren-1-yl)phosphine oxide (POPy2 ). The 31 P DNP-ssNMR spectra exhibited a sufficient signal-to-noise ratio to quantify the distribution of molecular orientations in amorphous films: the P=O axis of the vacuum-deposited and drop-cast POPy2 shows anisotropic and isotropic distribution, respectively. The different molecular orientations reflect the molecular origin of the different charge transport behaviors.
RESUMEN
Small molecule iron-chelators, siderophores, are very important in facilitating the acquisition of Fe(III), an essential element for pathogenic bacteria. Many Gram-negative outer-membrane transporters and Gram-positive lipoprotein siderophore-binding proteins have been characterized, and the binding ability of outer-membrane transporters and siderophore-binding proteins for Fe-siderophores has been determined. However, there is little information regarding the binding ability of these proteins for apo-siderophores, the iron-free chelators. Here we report that Bacillus cereus YxeB facilitates iron-exchange from Fe-siderophore to apo-siderophore bound to the protein, the first Gram-positive siderophore-shuttle system. YxeB binds ferrioxamine B (FO, Fe-siderophore)/desferrioxamine B (DFO, apo-siderophore) in vitro. Disc-diffusion assays and growth assays using the yxeB mutant reveal that YxeB is responsible for importing the FO. Cr-DFO (a FO analog) is bound by YxeB in vitro and B. cereus imports or binds Cr-DFO in vivo. In vivo uptake assays using Cr-DFO and FO and growth assays using DFO and Cr-DFO show that B. cereus selectively imports and uses FO when DFO is present. Moreover, in vitro competition assays using Cr-DFO and FO clearly demonstrate that YxeB binds only FO, not Cr-DFO, when DFO is bound to the protein. Iron-exchange from FO to DFO bound to YxeB must occur when DFO is initially bound by YxeB. Because the metal exchange rate is generally first order in replacement ligand concentration, protein binding of the apo-siderophore acts to dramatically enhance the iron exchange rate, a key component of the Gram-positive siderophore-shuttle mechanism.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Deferoxamina/metabolismo , Compuestos Férricos/metabolismo , Sideróforos/metabolismo , Proteínas Bacterianas/genética , Unión Competitiva , Transporte Biológico/fisiología , Cromatografía Líquida de Alta Presión , Pruebas Antimicrobianas de Difusión por Disco , Fluorescencia , Espectrometría de Masas , Plásmidos/genética , Unión Proteica , Análisis de RegresiónRESUMEN
Citrate is a common biomolecule that chelates Fe(III). Many bacteria and plants use ferric citrate to fulfill their nutritional requirement for iron. Only the Escherichia coli ferric citrate outer-membrane transport protein FecA has been characterized; little is known about other ferric citrate-binding proteins. Here we report a unique siderophore-binding protein from the gram-positive pathogenic bacterium Bacillus cereus that binds multinuclear ferric citrate complexes. We have demonstrated that B. cereus ATCC 14579 takes up (55)Fe radiolabeled ferric citrate and that a protein, BC_3466 [renamed FctC (ferric citrate-binding protein C)], binds ferric citrate. The dissociation constant (K(d)) of FctC at pH 7.4 with ferric citrate (molar ratio 1:50) is 2.6 nM. This is the tightest binding observed of any B. cereus siderophore-binding protein. Nano electrospray ionization-mass spectrometry (nano ESI-MS) analysis of FctC and ferric citrate complexes or citrate alone show that FctC binds diferric di-citrate, and triferric tricitrate, but does not bind ferric di-citrate, ferric monocitrate, or citrate alone. Significantly, the protein selectively binds triferric tricitrate even though this species is naturally present at very low equilibrium concentrations.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Compuestos Férricos/farmacocinética , Radioisótopos de Hierro/farmacocinética , Compuestos Férricos/metabolismo , Marcaje Isotópico , Espectrometría de Masas , Estructura Molecular , Unión Proteica , Sideróforos/metabolismoRESUMEN
Triarylboron compounds have attracted much attention, and found wide use as functional materials because of their electron-accepting properties arising from the vacant p orbitals on the boron atoms. In this study, we design and synthesize new donor-acceptor triarylboron emitters that show thermally activated delayed fluorescence. These emitters display sky-blue to green emission and high photoluminescence quantum yields of 87-100 % in host matrices. Organic light-emitting diodes using these emitting molecules as dopants exhibit high external quantum efficiencies of 14.0-22.8 %, which originate from efficient up-conversion from triplet to singlet states and subsequent efficient radiative decay from singlet to ground states.
RESUMEN
Dimers of partially oxygen-bridged triarylamines were designed and synthesized as hole-transporting materials. X-ray structural analyses revealed that these compounds form on-top π-stacking aggregates in the crystalline state. TRMC measurements showed that high levels of anisotropic charge transport were induced in the direction of the π-stacking. Surprisingly, even in vacuum-deposited amorphous films, these compounds retained some of the face-on π-stacking, thus facilitating an out-of-plane carrier mobility.
RESUMEN
Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Esterasas/química , Peptidoglicano/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Esterasas/genética , Esterasas/metabolismo , Eliminación de Gen , Cinética , Peptidoglicano/genética , Peptidoglicano/metabolismo , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genéticaRESUMEN
The YycG sensor histidine kinase co-ordinates cell wall remodelling with cell division in Gram-positive bacteria by controlling the transcription of genes for autolysins and their inhibitors. Bacillus subtilis YycG senses cell division and is enzymatically activated by associating with the divisome at the division septum. Here it is shown that the cytoplasmic PAS domain of this multi-domain transmembrane kinase is a determining factor translocating the kinase to the division septum. Furthermore, translocation to the division septum, per se, is insufficient to activate YycG, indicating that specific interactions and/or ligands produced there are required to stimulate kinase activity. N-terminal truncations of YycG lose negative regulation of their activity inferring that this regulation is accomplished through its transmembrane and extramembrane domains interacting with the membrane associated YycH and YycI proteins that do not localize to the divisome. The data indicate that YycG activity in non-dividing cells is suppressed by its interaction with YycH and YycI and its activation is co-ordinated to cell division in dividing cells by specific interactions that occur within the divisome.
Asunto(s)
Bacillus subtilis/fisiología , División Celular , Proteínas Quinasas/metabolismo , Histidina Quinasa , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Quinasas/genética , Eliminación de SecuenciaRESUMEN
For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-ß prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Muramidasa/fisiología , Peptidoglicano/química , Cationes , Cromatografía Líquida de Alta Presión/métodos , Endopeptidasas/química , Espectrometría de Masas/métodos , Muramidasa/química , Muramidasa/genética , Plásmidos/metabolismo , Polímeros/química , Polisacáridos/química , Profagos , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray/métodos , Zinc/químicaRESUMEN
The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimer's disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-ß (Aß) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/enzimología , Factor de Transcripción Activador 4/antagonistas & inhibidores , Factor de Transcripción Activador 4/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Quercetina/farmacología , Receptor Notch1/metabolismoRESUMEN
PURPOSE: In previous reports, it was demonstrated that bone marrow adipocytes were related to steroid osteoporosis through osteoclastogenesis induced by Receptor Activator of Nuclear factor κ-B Ligand (RANKL) expression. The purpose of this study was to evaluate the effect of Tumor necrosis factor-alpha (TNF-α) on RANKL expression in bone marrow adipocytes, and osteoclast differentiation supported by human bone marrow adipocytes. METHODS: RANKL, osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) mRNA expression in bone marrow adipocytes and their regulation by TNF-α treatment were measured by real-time RT-PCR. Co-cultures of bone marrow adipocytes and osteoclast precursors were performed with or without TNF-α, and osteoclast differentiation was evaluated morphologically and functionally. RESULTS: RANKL expression and an increase in the RANKL/OPG ratio in bone marrow adipocytes were stimulated by TNF-α treatment. In co-culture of bone marrow adipocytes and osteoclast precursors with TNF-α, the number of TRAP-positive multinuclear cells and resorption cavity formations of calcium phosphate film were increased. Osteoclast differentiation was suppressed by anti-RANKL antibody treatment. In co-culture with non-cell-contact conditions, no TRAP-positive cells or resorption cavity formations were observed. CONCLUSIONS: TNF-α increased RANKL expression in primary human bone marrow adipocytes. TNF-α induced the ability of bone marrow adipocytes to promote osteoclast differentiation and activity in a manner directly related to RANKL expression.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Osteoclastos/citología , Ligando RANK/genética , Factor de Necrosis Tumoral alfa/farmacología , Fosfatasa Ácida/metabolismo , Adipocitos/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Ligando RANK/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Coloración y Etiquetado , Fosfatasa Ácida Tartratorresistente , Factores de TiempoRESUMEN
BACKGROUND: Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1) is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins) against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. METHODS: Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years) undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. RESULTS: PAI-1 mRNA expression was up-regulated by 388% (P=0.002) with dexamethasone, and down-regulated by 45% (P=0.002) with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P=0.001) and simvastatin decreased total PAI-1 secretion by 64% (P=0.002). No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. CONCLUSION: The present study confirmed the suppressive effects of simvastatin on PAI-1 expression and secretion from bone marrow adipocytes. Furthermore, pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion. Simvastatin may thus exhibit preventive effects against steroid-induced osteonecrosis of the femoral head by suppressing PAI-1 secretion.
Asunto(s)
Adipocitos/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Dexametasona/toxicidad , Cuello Femoral/efectos de los fármacos , Glucocorticoides/toxicidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Simvastatina/farmacología , Adipocitos/metabolismo , Adipocitos/patología , Adiponectina/genética , Adiponectina/metabolismo , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Cuello Femoral/metabolismo , Cuello Femoral/patología , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
A profusion of phospholes: Diacenaphtho[1,2-b:1',2'-d]phospholes, a new class of arene-fused phosphole π-systems, were synthesized and their structural and electrochemical properties studied. The P-sulfide derivative has a high electron-transporting ability (µ(E) =2.4×10(-3) cm(2) V(-1) s(-1)) in a vacuum-deposited film.
RESUMEN
CwlQ (previous YjbJ) is one of the putative cell wall hydrolases in Bacillus subtilis. Its domain has an amino acid sequence similar to the soluble-lytic transglycosylase (SLT) of Escherichia coli Slt70 and also goose lysozyme (muramidase). To characterize the enzyme, the domain of CwlQ was cloned and expressed in E. coli. The purified CwlQ protein exhibited cell wall hydrolytic activity. Surprisingly, RP-HPLC, mass spectrometry (MS), and MS/MS analyses showed that CwlQ produces two products, 1,6-anhydro-N-acetylmuramic acid and N-acetylmuramic acid, thus indicating that CwlQ is a bifunctional enzyme. The site-directed mutagenesis revealed that glutamic acid 85 (Glu-85) is an amino acid residue essential to both activities.
Asunto(s)
Bacillus subtilis/enzimología , Glicosiltransferasas/química , Muramidasa/química , Dominio Catalítico/genética , Clonación Molecular , Escherichia coli/genética , Ácido Glutámico/química , Ácido Glutámico/genética , Glicosiltransferasas/genética , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de ProteínaRESUMEN
Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell-1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.
Asunto(s)
Técnicas Biosensibles , Shewanella , Proteínas Bacterianas/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Transporte de Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Shewanella/genética , Shewanella/metabolismoRESUMEN
Genetic circuits that encode extracellular electron transfer (EET) pathways allow the intracellular state of Escherichia coli to be electronically monitored and controlled. However, relatively low electron flux flows through these pathways, limiting the degree of control by these circuits. Since the EET pathway is composed of multiple multiheme cytochromes c (cyts c) from Shewanella oneidensis MR-1, we hypothesized that lower expression levels of cyt c may explain this low EET flux and may be caused by the differences in the cyt c maturation (ccm) machinery between these two species. Here, we constructed random mutations within ccmH by error-prone PCR and screened for increased cyt c production. We identified two ccmH mutants, ccmH-132 and ccmH-195, that exhibited increased heterologous cyt c expression, but had different effects on EET. The ccmH-132 strain reduced WO3 nanoparticles faster than the parental control, whereas the ccmH-195 strain reduced more slowly. The same trend is reflected in electrical current generation: ccmH-132, which has only a single mutation from WT, drastically increased current production by 77%. The percentage of different cyt c proteins in these two mutants suggests that the stoichiometry of the S. oneidensis cyts c is a key determinant of current production by Mtr-expressing E. coli. Thus, we conclude that modulating cyt c maturation effectively improves genetic circuits governing EET in engineered biological systems, enabling better bioelectronic control of E. coli.
Asunto(s)
Fuentes de Energía Bioeléctrica , Citocromos c/genética , Escherichia coli/genética , Ingeniería Genética/métodos , Shewanella/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Citocromos c/metabolismo , Electroquímica , Transporte de Electrón/genética , Electrones , Mutación , Nanopartículas/química , Operón , Óxidos/metabolismo , Tungsteno/metabolismoRESUMEN
Thermoelectric power generation from waste heat is an important component of future sustainable development. Ion-conducting materials are promising candidates because of their high Seebeck coefficients. This study demonstrates that ionic hydrogels based on imidazolium chloride salts exhibit outstanding Seebeck coefficients of up to 10 mV K-1. Along with their relatively high ionic conductivities (1.6 mS cm-1) and extremely low thermal conductivities (â¼0.2 W m-1 K-1), these hydrogels have good potential for use in heat recovery systems. The voltage behavior in response to temperature difference (stable or transient) differs significantly depending on the metal electrode material. We evaluated the electrode-dependent temperature sensitivity of the double layer capacitance of these hydrogels, which revealed that the thermally induced polarization of ions at the interface is one of the main contributors to the thermovoltage. Our results demonstrate the potential capability for ion and metal interactions to be used as an effective baseline for exploring ionic thermoelectric materials and devices. The developed thermoelectric supercapacitor exhibits reversible charging-discharging behavior under repeated disconnecting-connecting of an external load with a constant temperature difference, which offers a novel strategy for heat-to-electricity energy conversion from steady-temperature heat sources.