RESUMEN
Biophotovoltaic devices utilize photosynthetic organisms such as the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) to generate current for power or hydrogen production from light. These devices have been improved by both architecture engineering and genetic engineering of the phototrophic organism. However, genetic approaches are limited by lack of understanding of cellular mechanisms of electron transfer from internal metabolism to the cell exterior. Type IV pili have been implicated in extracellular electron transfer (EET) in some species of heterotrophic bacteria. Furthermore, conductive cell surface filaments have been reported for cyanobacteria, including Synechocystis. However, it remains unclear whether these filaments are type IV pili and whether they are involved in EET. Herein, a mediatorless electrochemical setup is used to compare the electrogenic output of wild-type Synechocystis to that of a ΔpilD mutant that cannot produce type IV pili. No differences in photocurrent, i.e., current in response to illumination, are detectable. Furthermore, measurements of individual pili using conductive atomic force microscopy indicate these structures are not conductive. These results suggest that pili are not required for EET by Synechocystis, supporting a role for shuttling of electrons via soluble redox mediators or direct interactions between the cell surface and extracellular substrates.
RESUMEN
Cyanovirin-N (CVN), a cyanobacterial lectin, exemplifies a class of antiviral agents that inhibit HIV by binding to the highly glycosylated envelope protein gp120. Here, we investigate the energetics of glycan recognition using a computationally inexpensive flexible docking approach, backbone perturbation docking (BP-Dock). We benchmarked our method using two mutants of CVN: P51G-m4-CVN, which binds dimannose with high affinity through domain B, and CVN((mutDB)), in which binding to domain B has been abolished through mutation of five polar residues to small nonpolar side chains. We investigated the energetic contribution of these polar residues along with the additional position 53 by docking dimannose to single-point CVN mutant models. Analysis of the docking simulations indicated that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen-bond network that reverberated throughout the binding cavity. N42A decreased the binding score to a level comparable to that of CVN((mutDB)) by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score, similar to P51G-m4-CVN. Experimental characterization of the five mutants by NMR spectroscopy confirmed the binding affinity pattern predicted by BP-Dock. Despite their mostly conserved fold and stability, E41A, E41G, and T57A displayed dissociation constants in the millimolar range. N53S showed a binding constant in the low micromolar range, similar to that observed for P51G-m4-CVN. No binding was observed for N42A. Our results show that BP-Dock is a useful tool for rapidly screening the relative binding affinity pattern of in silico-designed mutants compared with wild-type, supporting its use to design novel mutants with enhanced binding properties.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Simulación del Acoplamiento Molecular , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
Biophotovoltaic devices employ photosynthetic organisms at the anode of a microbial fuel cell to generate electrical power. Although a range of cyanobacteria and algae have been shown to generate photocurrent in devices of a multitude of architectures, mechanistic understanding of extracellular electron transfer by phototrophs remains minimal. Here we describe a mediatorless bioelectrochemical device to measure the electrogenic output of a planktonically grown cyanobacterium, Synechocystis sp. PCC6803. Light dependent production of current is measured, and its magnitude is shown to scale with microbial cell concentration and light intensity. Bioelectrochemical characterization of a Synechocystis mutant lacking Photosystem II demonstrates conclusively that production of the majority of photocurrent requires a functional water splitting aparatus and electrons are likely ultimately derived from water. This shows the potential of the device to rapidly and quantitatively characterize photocurrent production by genetically modified strains, an approach that can be used in future studies to delineate the mechanisms of cyanobacterial extracellular electron transport.
Asunto(s)
Transporte de Electrón , Espacio Extracelular/metabolismo , Synechocystis/metabolismo , Electroquímica , Oxidación-Reducción , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of casein (used as a fining agent) present in white wines. Although minute amounts (200 microL) of CPLL beads are added to the entire content of a wine bottle (750 mL), they are able to sequester with high efficiency (up to 80%) residual traces of casein, permitting a signal "amplification" of at least 5000-fold. It is here demonstrated that as little as 1 microg/L of casein can be efficiently detected in white wines, a major improvement over previous investigations in which the lower detection limit had been estimated at 100 microg/L. The fact that such very low levels of fining agents can still be detected in treated white wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.
Asunto(s)
Caseínas/análisis , Aditivos Alimentarios/análisis , Proteómica/métodos , Vino/análisis , Animales , Bovinos , Límite de Detección , Biblioteca de PéptidosRESUMEN
The tandem domain rhodanese-homology protein RhdA of Azotobacter vinelandii shows an active-site loop structure that confers structural peculiarity in the environment of its catalytic cysteine residue. The in vivo effects of the lack of RhdA were investigated using an A. vinelandii mutant strain (MV474) in which the rhdA gene was disrupted by deletion. Here, by combining analytical measurements and transcript profiles, we show that deletion of the rhdA gene generates an oxidative stress condition to which A. vinelandii responds by activating defensive mechanisms. In conditions of growth in the presence of the superoxide generator phenazine methosulfate, a stressor-dependent induction of rhdA gene expression was observed, thus highlighting that RhdA is important for A. vinelandii to sustain oxidative stress. The potential of RhdA to buffer general levels of oxidants in A. vinelandii cells via redox reactions involving its cysteine thiol is discussed.
Asunto(s)
Azotobacter vinelandii/enzimología , Tiosulfato Azufretransferasa/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Dominio Catalítico , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Tiosulfato Azufretransferasa/químicaRESUMEN
The hemolymph of Limulus polyphemus, a very ancient marine arthropod dating back to ca. 440 million years, has been explored in depth via capture by combinatorial peptide ligand libraries. Whereas barely a dozen proteins had been known up to the present, we have increased this number by more than 1 order of magnitude, up to 160 unique gene products, identified via the dbEST_limulus as well as via comparison with the other members of the Chelicerata subphylum to which Limulus belongs, namely, scorpions, ticks, mites, and spiders. Yet we have sequences of many other peptides, suggesting the presence of at least one more order of magnitude of species (1000 and more), that could not be identified as such sequences have no counterparts in present databases. This further reinforces the notion that these could be ancestral proteins, scarcely represented in present times. These data might represent the true birth of paleo-proteomics.
Asunto(s)
Hemolinfa/química , Cangrejos Herradura/química , Biblioteca de Péptidos , Proteómica/métodos , Animales , Transporte Biológico , Técnicas Químicas Combinatorias/métodos , Electroforesis en Gel Bidimensional , Femenino , Redes y Vías Metabólicas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteoma/química , Proteoma/metabolismo , Especificidad de la Especie , Tripsina/metabolismoRESUMEN
The rhdA gene of Azotobacter vinelandii codes for RhdA, a rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe-S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the present study we analysed the defects of the rhdA-null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner-Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe-4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys(230) thiol.
Asunto(s)
Azotobacter vinelandii/metabolismo , Estrés Oxidativo , Tiosulfato Azufretransferasa/deficiencia , Tiosulfato Azufretransferasa/metabolismo , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Carbono/metabolismo , Gluconatos/metabolismo , Viabilidad Microbiana , Mutación/genética , Oxidación-Reducción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Superóxidos/metabolismo , Tiosulfato Azufretransferasa/química , Tiosulfato Azufretransferasa/genéticaRESUMEN
In Azotobacter vinelandii the rhdA gene codes for a protein (RhdA) of the rhodanese-homology superfamily. By combining proteomics, enzymic profiles and ultrastructural observations, the phenotype of an A. vinelandii rhdA mutant was analyzed. We found that the A. vinelandii rhdA mutant, and not the wild-type strain, accumulated polyhydroxybutyrate. RhdA deficiency enhanced the expression of enzymes of the polyhydroxybutyrate biosynthetic operon, and affected the activity of specific tricarboxylic acid cycle enzymes. The effect was dramatic on aconitase, in spite of comparable expression of aconitase polypeptides in both strains. By using a model system, we found that RhdA triggered protection from oxidants.
Asunto(s)
Azotobacter vinelandii/enzimología , Proteínas Bacterianas/fisiología , Estrés Oxidativo , Tiosulfato Azufretransferasa/fisiología , Azotobacter vinelandii/genética , Azotobacter vinelandii/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Electroforesis en Gel Bidimensional , Genes Bacterianos , Metosulfato de Metilfenazonio/farmacología , Mutación , Oxidantes/farmacología , Estrés Oxidativo/genética , Fenotipo , Proteómica , Tiosulfato Azufretransferasa/deficiencia , Tiosulfato Azufretransferasa/genéticaRESUMEN
After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.
Asunto(s)
Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/metabolismo , Azufre/química , Tiosulfato Azufretransferasa/biosíntesis , Tiosulfato Azufretransferasa/metabolismo , Azotobacter vinelandii/enzimología , Dominio Catalítico , Cisteína/química , Escherichia coli/genética , Histidina/química , Mapeo Peptídico , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Azufre/metabolismo , Tiosulfato Azufretransferasa/química , Tiosulfato Azufretransferasa/genética , Tiosulfato Azufretransferasa/aislamiento & purificaciónRESUMEN
The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.