RESUMO
Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.
Assuntos
Escherichia coli/enzimologia , Hidroquinonas/química , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Vitamina K 2/análogos & derivados , Benzoquinonas/metabolismo , Respiração Celular , Espectroscopia de Ressonância de Spin Eletrônica , Hidroquinonas/metabolismo , Cinética , Naftóis/química , Oxirredução , Vitamina K 2/química , Vitamina K 2/metabolismoRESUMO
Abiotic and biotic processes associated with the degradation of a light petroleum in brines close to the salt-saturation (~31 %) and the effect of labile organic matter (LOM) supply (casaminoacids/citrate; 0.2 and 0.1 % w/v, respectively) were followed during an incubation of 30 days. After 4-week incubation at 40 °C under light/dark cycles, a 24 % of abiotic degradation was observed in untreated brines. The stimulation of native brines community with LOM addition allowed an additional 12.8 % oil attenuation due to biodegradation processes. Successional changes in the active microbial community structure due to the oil contamination (16S rRNA DGGE approach) showed the selection of one phylotype affiliated to Salinibacter and the disappearance of Haloquadratum walsbyi in untreated brines. In LOM-amended microcosms, phylotypes related to Salinibacter, Haloarcula, Haloterrigena and Halorhabdus were selected. An effect of hydrocarbon contamination was only observed in the bacterial community with the inhibition of two dominant proteobacterial phylotypes. This study further confirms that short-term and moderate oil biodegradation is possible in LOM-stimulated brines. Biodegradation should be much more reduced under in situ conditions. Self-cleaning capacities of close to saturation hypersaline lakes appears, therefore very limited compared to non-extreme haline environments.
Assuntos
Recuperação e Remediação Ambiental , Lagos/microbiologia , Microbiota , Petróleo/microbiologia , Salinidade , Halobacteriales/genética , Halobacteriales/isolamento & purificação , Halobacteriales/metabolismo , Lagos/química , Petróleo/metabolismo , Poluição por Petróleo , SaisRESUMO
Martinique's mangroves, which cover 1.85 ha of the island (<0.1 % of the total area), are considerably vulnerable to local urban, agricultural, and industrial pollutants. Unlike for temperate ecosystems, there are limited indicators that can be used to assess the anthropogenic pressures on mangroves. This study investigated four stations on Martinique Island, with each being subject to varying anthropogenic pressures. An analysis of mangrove sediment cores approximately 18 cm in depth revealed two primary types of pressures on Martinique mangroves: (i) an enrichment in organic matter in the two stations within the highly urbanized bay of Fort-de-France and (ii) agricultural pressure observed in the four studied mangrove stations. This pressure was characterized by contamination, exceeding the regulatory thresholds, with dieldrin, total DDT, and metals (As, Cu and Ni) found in phytosanitary products. The mangroves of Martinique are subjected to varying degrees of anthropogenic pressure, but all are subjected to contamination by organochlorine pesticides. Mangroves within the bay of Fort-de-France experience notably higher pressures compared to those in the island's northern and southern regions. In these contexts, the microbial communities exhibited distinct responses. The microbial biomass and the abundance of bacteria and archaea were higher in the two less-impacted stations, while in the mangrove of Fort-de-France, various phyla typically associated with polluted environments were more prevalent. These differences in the microbiota composition led to the identification of 65 taxa, including Acanthopleuribacteraceae, Spirochaetaceae, and Pirellulaceae, that could potentially serve as indicators of an anthropogenic influence on the mangrove sediments of Martinique Island.
Assuntos
Monitoramento Ambiental , Áreas Alagadas , Monitoramento Ambiental/métodos , Martinica , Agricultura , Poluentes Químicos da Água/análise , Sedimentos Geológicos/microbiologia , Sedimentos Geológicos/química , MicrobiotaRESUMO
Mangroves develop under environmental conditions and anthropogenic pressures whose impact on benthic meiofauna remains poorly understood. It is unclear how meiofauna communities are structured according to local sedimentary conditions. This study was designed to characterize the community structure of meiofauna and nematodes (dominant taxa) and the associated environmental forcings in intertidal mangrove sediments from Mayotte (Indo-West-Pacific), Martinique and Guadeloupe (Caribbean). Sediment cores were sampled at the end of the dry season at low tide on adult mangrove stands with similar immersion time. In each sediment layer, we analyzed redox potential, pH, porewater salinity, grain size, organic matter, metals, organic contaminants, prokaryotes and meiofauna. Our results show that sediments far from cities and agricultural fields trapped site-specific contaminants due to local water transport processes. Some metals, PAHs or pesticides exceeded toxicity thresholds in most of the studied stations, thus being harmful to benthic fauna. The sedimentary environment acts as a filter selecting specific meiofauna communities at station scale only in the Caribbean. In Mayotte, horizontal homogeneity contrasts with vertical heterogeneity of the sedimentary environment and the meiofauna. Nematode genera showed particular distribution patterns horizontally and vertically, suggesting the presence of sediment patches suitable for a restricted pool of genera on each island. Results in the Caribbean are consistent with nested diversity patterns due to environmental filtering. Conversely, horizontal homogeneity at Mayotte would reflect greater dispersal between stations or more spatially homogeneous anthropogenic pressures. The nematode genera present at depth may not be the most specialized, but the most versatile, capable of thriving in different conditions. Terschellingia and Daptonema showed contrasted responses to environmental forcing, likely due to their versatility, while Desmodora showed uniform responses between study areas, except when toxicity thresholds were exceeded. Our results emphasize that a given genus of nematode may respond differently to sedimentary conditions depending on sites.
Assuntos
Monitoramento Ambiental , Sedimentos Geológicos , Nematoides , Áreas Alagadas , Animais , Sedimentos Geológicos/química , Poluentes Químicos da Água/análise , Região do Caribe , Guadalupe , InvertebradosRESUMO
The microbial communities inhabiting the Atlantic-East Pacific (AEP) mangroves have been poorly studied, and mostly comprise chronically polluted mangroves. In this study, we characterized changes in the structure and diversity of microbial communities of mangroves along the urban-to-rural gradient of the Cayenne estuary (French Guiana, South America) that experience low human impact. The microbial communities were assigned into 50 phyla. Proteobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, and Planctomycetes were the most abundant taxa. The environmental determinants found to significantly correlated to the microbial communities at these mangroves were granulometry, dieldrin concentration, pH, and total carbon (TC) content. Furthermore, a precise analysis of the sediment highlights the existence of three types of anthropogenic pressure among the stations: (i) organic matter (OM) enrichment due to the proximity to the city and its wastewater treatment plant, (ii) dieldrin contamination, and (iii) naphthalene contamination. These forms of weak anthropogenic pressure seemed to impact the bacterial population size and microbial assemblages. A decrease in Bathyarchaeota, "Candidatus Nitrosopumilus", and Nitrospira genera was observed in mangroves subjected to OM enrichment. Mangroves polluted with organic contaminants were enriched in Desulfobacteraceae, Desulfarculaceae, and Acanthopleuribacteraceae (with dieldrin or polychlorobiphenyl contamination), and Chitinophagaceae and Geobacteraceae (with naphthalene contamination). These findings provide insights into the main environmental factors shaping microbial communities of mangroves in the AEP that experience low human impact and allow for the identification of several potential microbial bioindicators of weak anthropogenic pressure.
Assuntos
Microbiota , Efeitos Antropogênicos , Biomarcadores Ambientais , Estuários , Guiana Francesa , Sedimentos Geológicos , Humanos , Planctomicetos , Áreas AlagadasRESUMO
Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Espaço Intracelular/metabolismo , Complexos Multienzimáticos/metabolismo , Nitrato Redutase/metabolismo , Consumo de Oxigênio/fisiologia , Fracionamento Celular , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Nitrato Redutase/genética , Plasmídeos/genética , Subunidades Proteicas/metabolismo , Estatísticas não ParamétricasRESUMO
To study the impact of oxygen regimes on the removal of polycylic aromatic hydrocarbons (PAHs) in oil-spill-affected coastal marine sediments, we used a thin-layer incubation method to ensure that the incubated sediment was fully oxic, anoxic, or was influenced by oxic-anoxic switches without sediment stirring. Hydrocarbon content and microbial assemblages were followed during 60 days to determine PAH degradation kinetics and microbial community dynamics according to the oxygenation regimes. The highest PAH removal, with 69 % reduction, was obtained at the end of the experiment under oxic conditions, whereas weaker removals were obtained under oscillating and anoxic conditions (18 and 12 %, respectively). Bacterial community structure during the experiment was determined using a dual 16S rRNA genes/16S rRNA transcripts approach, allowing the characterization of metabolically active bacteria responsible for the functioning of the bacterial community in the contaminated sediment. The shift of the metabolically active bacterial communities showed that the selection of first responders belonged to Pseudomonas spp. and Labrenzia sp. and included an unidentified Deltaproteobacteria-irrespective of the oxygen regime-followed by the selection of late responders adapted to the oxygen regime. A novel unaffiliated phylotype (B38) was highly active during the last stage of the experiment, at which time, the low-molecular-weight (LMW) PAH biodegradation rates were significant for permanent oxic- and oxygen-oscillating conditions, suggesting that this novel phylotype plays an active role during the restoration phase of the studied ecosystem.
Assuntos
Bactérias , Sedimentos Geológicos/microbiologia , Hidrocarbonetos/metabolismo , Oxigênio/metabolismo , Poluição por Petróleo , Poluentes Químicos da Água/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Ecossistema , Sedimentos Geológicos/química , Hidrocarbonetos/análise , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Poluentes Químicos da Água/análiseRESUMO
Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.