Molecular evidence for stimulation of methane oxidation in Amazonian floodplains by ammonia-oxidizing communities.

Ammonia oxidation is the rate-limiting first step of nitrification and a key process in the nitrogen cycle that results in the formation of nitrite (NO2 -), which can be further oxidized to nitrate (NO3 -). In the Amazonian floodplains, soils are subjected to extended seasons of flooding during the rainy season, in which they can become anoxic and produce a significant amount of methane (CH4). Various microorganisms in this anoxic environment can couple the reduction of different ions, such as NO2 - and NO3 -, with the oxidation of CH4 for energy production and effectively link the carbon and nitrogen cycle. Here, we addressed the composition of ammonium (NH4 +) and NO3 --and NO2 --dependent CH4-oxidizing microbial communities in an Amazonian floodplain. In addition, we analyzed the influence of environmental and geochemical factors on these microbial communities. Soil samples were collected from different layers of forest and agroforest land-use systems during the flood and non-flood seasons in the floodplain of the Tocantins River, and next-generation sequencing of archaeal and bacterial 16S rRNA amplicons was performed, coupled with chemical characterization of the soils. We found that ammonia-oxidizing archaea (AOA) were more abundant than ammonia-oxidizing bacteria (AOB) during both flood and non-flood seasons. Nitrogen-dependent anaerobic methane oxidizers (N-DAMO) from both the archaeal and bacterial domains were also found in both seasons, with higher abundance in the flood season. The different seasons, land uses, and depths analyzed had a significant influence on the soil chemical factors and also affected the abundance and composition of AOA, AOB, and N-DAMO. During the flood season, there was a significant correlation between ammonia oxidizers and N-DAMO, indicating the possible role of these oxidizers in providing oxidized nitrogen species for methanotrophy under anaerobic conditions, which is essential for nitrogen removal in these soils.

Methane emission suppression in flooded soil from Amazonia.

The coupling between ferrous iron and methane production has important global implications, with iron ions acting as electron acceptors for anaerobic oxidation of methane (AOM) and inhibitors of methanogenesis in different environments, including floodplain soils. In this sense, we analyzed the relationship between Fe(II) concentration and methane production in soil layers collected at 0-15 cm and 15-30 cm from flooded-forest and -agroforestry in Amazonian clear water floodplain incubated in anaerobic batch reactors using acetate, formate and glucose as organic sources. High throughput sequencing of archaeal and bacterial 16S rRNA genes was employed to assess the abundance and composition of the active methanogenic and methanotrophic microbial groups potentially involved in Fe(III)-dependent AOM in the soil used as inoculum. Positive correlation was revealed between Fe(II) concentration and methane production, with higher accumulation of Fe(II) in incubated soil layer collected at 0-15 cm in both forest and agroforestry sites for all the three organic sources. The accumulation of Fe(II) in the incubated soil evidenced the oxidation of Fe(III) potentially by Methanobacterium, Desulfobulbus and 'Candidatus methanoperedens nitroreducens' living in anaerobic condition at this soil layer. The results point out to the microbial ferric iron reduction as an important potential pathway for anaerobic organic matter decomposition in Amazonian floodplain, evidencing methanogenesis suppression by Fe(III) reduction in flooded-forest and -agroforestry in Amazonian clear water river floodplain.

MeCP2 Deficiency Disrupts Kainate-Induced Presynaptic Plasticity in the Mossy Fiber Projections in the Hippocampus.

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases.

Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

Metaproteomics reveals metabolic transitions between healthy and diseased stony coral Mussismilia braziliensis.

Infectious diseases such as white plague syndrome (WPS) and black band disease (BBD) have caused massive coral loss worldwide. We performed a metaproteomic study on the Abrolhos coral Mussismilia braziliensis to define the types of proteins expressed in healthy corals compared to WPS- and BBD-affected corals. A total of 6363 MS/MS spectra were identified as 361 different proteins. Healthy corals had a set of proteins that may be considered markers of holobiont homoeostasis, including tubulin, histone, Rab family, ribosomal, peridinin-chlorophyll a-binding protein, F0F1-type ATP synthase, alpha-iG protein, calmodulin and ADP-ribosylation factor. Cnidaria proteins found in healthy M. braziliensis were associated with Cnidaria-Symbiodinium endosymbiosis and included chaperones (hsp70, hsp90 and calreticulin), structural and membrane modelling proteins (actin) and proteins with functions related to intracellular vesicular traffic (Rab7 and ADP-ribosylation factor 1) and signal transduction (14-3-3 protein and calmodulin). WPS resulted in a clear shift in the predominance of proteins, from those related to aerobic nitrogen-fixing bacteria (i.e. Rhizobiales, Sphingomonadales and Actinomycetales) in healthy corals to those produced by facultative/anaerobic sulphate-reducing bacteria (i.e. Enterobacteriales, Alteromonadales, Clostridiales and Bacteroidetes) in WPS corals. BBD corals developed a diverse community dominated by cyanobacteria and sulphur cycle bacteria. Hsp60, hsp90 and adenosylhomocysteinase proteins were produced mainly by cyanobacteria in BBD corals, which is consistent with elevated oxidative stress in hydrogen sulphide- and cyanotoxin-rich environments. This study demonstrates the usefulness of metaproteomics for gaining better comprehension of coral metabolic status in health and disease, especially in reef systems such as the Abrolhos that are suffering from the increase in global and local threatening events.

Extracellular vesicle-mediated export of fungal RNA.

Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. In this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. The EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis.

Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location.

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor.

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.