Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Integrative transcriptome and whole-genome bisulfite sequencing analyses of a temperature-sensitive albino tea plant cultivar.

Green tea made from albino buds and leaves has a strong umami taste and aroma. The cultivar 'Zhonghuang 2' (ZH2, Camellia sinensis) is a natural mutant with young shoots that are yellow in spring and green or yellow-green in summer. However, the mechanism of leaf color change remains unclear. Here, we found that young shoots of ZH2 were yellow at low temperature (LT) and green at high temperature (HT), indicating that ZH2 is a temperature-sensitive cultivar. Transmission electron microscopy analysis showed that the grana in the chloroplasts of young shoots grown at LT were poorly stacked, which caused a lack of photoreactions and chlorophyll. RNA-seq results showed 1279 genes differentially expressed in the young shoots grown at LT compared with those at HT, including genes related to cytochrome synthesis, chloroplast development, photosynthesis, and DNA methylation. A whole-genome bisulfite sequencing assay revealed that the dynamics of DNA methylation levels in the CG, CHG, and CHH contexts decreased under LT, and the change was most obvious in the CHH context. Furthermore, 72 genes showed significant changes in both expression and DNA methylation levels, and most of them were related to cytochrome synthesis, chloroplast development, photosynthesis, transcription factors, and signaling pathways. These results demonstrate that DNA methylation is involved in the LT-regulated albino processes of ZH2. Changes in DNA methylation levels were associated with changes in gene expression levels, affecting the structure and function of chloroplasts, which may have a phenotypic impact on shoot and leaf color.

Transcriptomic insights unveil the crucial roles of cytochromes, NADH, and pili in Ag(I) reduction by Geobacter sulfurreducens.

Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens, offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c-type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH's potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction.

Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Morphological, molecular characterization and taxonomic status of Triplophysa marmorata and Triplophysa kashmirensis (Cypriniformes: Nemacheilidae) from Kashmir valley, India

AbstractIn India the distribution of genus Triplophysa has been reported only in the upper drainage of the Indus River in Jammu and Kashmir and Lahul and Spiti area of Himachal Pradesh. There is no study on the taxonomic characterization of this genus from Kashmir Himalaya. Therefore the present study was aimed to characterize two important fish species Triplophysa marmorata and T. kashmirensis from Kashmir valley, by using morphometric and molecular tools. It is difficult to discriminate these two species due to the poor quality of original descriptions, and the lack of good reviews. Keeping this in view, a morphometric and molecular study was conducted. Morphometric data were analyzed by using univariate analysis of variance (ANOvA) and multivariate analyses (Principal component analysis) and mtDNA marker Cytochrome oxidase 1 was used for molecular support. Altogether, 22 morphometric characters were used and 15 characters were found significantly variable (P < 0.05). First two components of principal component analysis (PCA) i.e. PC1 and PC2 grouped these two species into separate clusters. The Cytochrome oxidase 1 analysis showed that the mean intraspecific nucleotide divergence (K2P) was 0.001 and interspecific nucleotide divergence was 0.007. Despite having low K2P divergence, these two species got separated into two distinct clades in both Neighbour joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree building methods. But the pattern of clade formation showed that these species were recently radiated from each other and may have the same ancestor. Furthermore, these two species were found closer to Nemacheilidae than to Balitoridae family in the phylogenetic analysis. The molecular divergence between these species was also supported by variance in morphometric data. This work may build the base for the revision of taxonomic identity of these two important fishes of genus Triplophysa. The present investigation formulated that, based on morphological and mtDNA COI sequences analysis, these two taxonomic Triplophysa species should be considered as valid. The results may further assist to enhance the knowledge of the ichthyologists in understanding the ichthyofauna of Kashmir valley and will help them in planning strategies for conservation and management of these less studied small indigenous species along their natural range of distribution. Rev. Biol. Trop. 64 (2): 473-482. Epub 2016 June 01.

ResumenEn la India, la distribución del género Triplophysa se ha reportado solo en la parte superior del río Indus en Jammu, Kashmir, Lahul y Spiti en el área de Himachal Pradesh. No existen publicaciones acerca de la caracterización taxonómica de este género en Kashmir Himalaya. Por lo tanto, en este estudio se caracterizaron dos especies del valle de Kashmir: Triplophysa marmorata y T. kashmirensi, mediante el uso de herramientas morfométricas y moleculares. Es difícil diferenciar entre estas dos species debido a las vagas descripciones originales y a la falta de buenas revisiones. Debido a esto se realizó un estudio morfométrico y molecular. Los datos morfométricos se analizaron mediante un ANOvA y un análisis de componentes principales y el marcador del gen de la citocromo oxidasa 1 se usó para apoyo molecular. En general, se usaron 22 caracteres morfométricos y 15 fueron significativos (P < 0.05). Los dos primeros componentes del análisis de components principales (PCA), PC1 y PC2, agruparon estas dos especies en clusters separados. El análisis con citocromo oxidasa 1 mostró que el promedio de divergencia del nucleótido intraespecífico (K2P) fue de 0.001 y la divergencia del nucleótido intraespecífico fue de 0.007. A pesar de la baja divergencia de K2P, estas dos species se separan en dos clados diferentes tanto por el método NJ como por el método UPGMA. Sin embargo, el patrón de formación del clado mostró que estas species radiaron recientemente una de la otra y que podrían tener un ancestro en común. Además, en el análisis filogenético estas dos especies se encontraron más cerca de la familia Nemacheilidae que de Balitoridae. La divergencia molecular entre estas dos especies también fue respaldada por la varianza en los datos morfométricos. Este estudio puede establecer la base para una revisión taxonómica de estos dos importantes peces del género Triplophysa. Esta investigación postuló que, basada en análisis morfológicos y de secuencia de ADNm COI, estas dos especies taxonómicas de Triplophysa deben considerarse válidas. Los resultados pueden contribuir a mejorar el conocimiento de los ictiólogos en la comprensión de la ictiofauna del valle de Kashmir y les ayudará a planear estrategias de conservación y manejo de estas dos pequeñas especies indígenas y poco estudiadas en su rango de distribución natural.

Ultrastructural and metabolic alteration in dyskinetoplastic Trypanosoma cruzi

El cultivo del Trypanosoma cruzi (agente causal de la enfermedad de Chagas; Kinetoplstidae; cepa Tulahuen) en presencia de etidio, una droga de efecto selectivos sobre el DNA mitocondrial, produjo la inhibición del crecimiento del parásito (50% con 1 µg/ml de droga) y alteraciones típicas en el DNA cinetoplástico, verificadas por microscopía electrónica. Los kinetoplastos se vuelven irregulares y erráticos en la ubicación, pierden la forma de arco y las diferencias de las membranas (densidad y crestas), la matriz se vuelve floculenta y filamentosa y el k DNA se compacta y pierde la configuración fibrilar. La espectrofotometría diferencial de los epimastigotes discinetoplásticos demostró la disminución de los citocromos aa3, b y o, mientras que el citocromo c no fue afectado. El efecto del etidio fue maximo sobre aa3; mínimo sobre o y en todos los casos proporcional a la concentración de la droga. Con 1 µg/ml de bromuro de etidio la disminución del citocromo aa3 fue de 93% (promedio de 4 determinaciones ñ E.S.). La respuesta de los epimastigotes discinetoplásticos a los inhibidores de la cadena respiratoria mitocondrial, a saber, cianuro (concentraciones hasta 200 µM), azida y antimicina fue negativa, en contraste con los epismatigotes normales, cuya respiración fue inhibida 75-85% por los mismos inhibidores. El cianuro 1-3 mM inhibió, sin embargo, la respiración endógena de los epimastigotes discineto-plasticos y también la respiración antimicina o azida-insensible de los epimastigotes normales. Resultados similares se obtuvieron con la fracción mitocondrial de T. cruzi, que contiene el citocromo o. Teniendo en cuenta esta circunstancia se postula la operación de una xidasa terminal accesoria, inhibida por el cianuro en concentraciones relativamente altas. El ácido salicihidromáxico, un inhibidor de la respiración dependiente de la oxidasa Cu-dependiente de otros tripanosomatideos, no afectó la respiración del T. cruzi normal o discinetoplástico