Probing Denitrifying Anaerobic Methane Oxidation via Antimicrobial Intervention: Implications for Innovative Wastewater Management.

Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO) has the potential for application in wastewater treatment plants. However, our understanding of the N-DAMO process is primarily based on studies conducted on environmental samples or enrichment cultures using metagenomic approaches. To gain deeper insights into N-DAMO, we used antimicrobial compounds to study the function and physiology of 'Candidatus Methanoperedens nitroreducens' and 'Candidatus Methylomirabilis oxyfera' in N-DAMO enrichment cultures. We explored the effects of inhibitors and antibiotics and investigated the potential application of N-DAMO in wastewater contaminated with ammonium and heavy metals. Our results showed that 'Ca. M. nitroreducens' was susceptible to puromycin and 2-bromoethanesulfonate, while the novel methanogen inhibitor 3-nitrooxypropanol had no effect on N-DAMO. Furthermore, 'Ca. M. oxyfera' was shown to be susceptible to the particulate methane monooxygenase inhibitor 1,7-octadiyne and a bacteria-suppressing antibiotic cocktail. The N-DAMO activity was not affected by ammonium concentrations below 10 mM. Finally, the N-DAMO community appeared to be remarkably resistant to lead (Pb) but susceptible to nickel (Ni) and cadmium (Cd). This study provides insights into microbial functions in N-DAMO communities, facilitating further investigation of their application in methanogenic, nitrogen-polluted water systems.

Sulfide Toxicity as Key Control on Anaerobic Oxidation of Methane in Eutrophic Coastal Sediments.

Coastal zones account for 75% of marine methane emissions, despite covering only 15% of the ocean surface area. In these ecosystems, the tight balance between methane production and oxidation in sediments prevents most methane from escaping into seawater. However, anthropogenic activities could disrupt this balance, leading to an increased methane escape from coastal sediments. To quantify and unravel potential mechanisms underlying this disruption, we used a suite of biogeochemical and microbiological analyses to investigate the impact of anthropogenically induced redox shifts on methane cycling in sediments from three sites with contrasting bottom water redox conditions (oxic-hypoxic-euxinic) in the eutrophic Stockholm Archipelago. Our results indicate that the methane production potential increased under hypoxia and euxinia, while anaerobic oxidation of methane was disrupted under euxinia. Experimental, genomic, and biogeochemical data suggest that the virtual disappearance of methane-oxidizing archaea at the euxinic site occurred due to sulfide toxicity. This could explain a near 7-fold increase in the extent of escape of benthic methane at the euxinic site relative to the hypoxic one. In conclusion, these insights reveal how the development of euxinia could disrupt the coastal methane biofilter, potentially leading to increased methane emissions from coastal zones.

Microbial degradation of plant toxins.

Plants produce a variety of secondary metabolites in response to biotic and abiotic stresses. Although they have many functions, a subclass of toxic secondary metabolites mainly serve plants as deterring agents against herbivores, insects, or pathogens. Microorganisms present in divergent ecological niches, such as soil, water, or insect and rumen gut systems have been found capable of detoxifying these metabolites. As a result of detoxification, microbes gain growth nutrients and benefit their herbivory host via detoxifying symbiosis. Here, we review current knowledge on microbial degradation of toxic alkaloids, glucosinolates, terpenes, and polyphenols with an emphasis on the genes and enzymes involved in breakdown pathways. We highlight that the insect-associated microbes might find application in biotechnology and become targets for an alternative microbial pest control strategy.

Anaerobic methanotrophy is stimulated by graphene oxide in a brackish urban canal sediment.

Anthropogenic activities are influencing aquatic environments through increased chemical pollution and thus are greatly affecting the biogeochemical cycling of elements. This has increased greenhouse gas emissions, particularly methane, from lakes, wetlands, and canals. Most of the methane produced in anoxic sediments is converted into carbon dioxide by methanotrophs before it reaches the atmosphere. Anaerobic oxidation of methane requires an electron acceptor such as sulphate, nitrate, or metal oxides. Here, we explore the anaerobic methanotrophy in sediments of three urban canals in Amsterdam, covering a gradient from freshwater to brackish conditions. Biogeochemical analysis showed the presence of a shallow sulphate-methane transition zone in sediments of the most brackish canal, suggesting that sulphate could be a relevant electron acceptor for anaerobic methanotrophy in this setting. However, sediment incubations amended with sulphate or iron oxides (ferrihydrite) did not lead to detectable rates of methanotrophy. Despite the presence of known nitrate-dependent anaerobic methanotrophs (Methanoperedenaceae), no nitrate-driven methanotrophy was observed in any of the investigated sediments either. Interestingly, graphene oxide stimulated anaerobic methanotrophy in incubations of brackish canal sediment, possibly catalysed by anaerobic methanotrophs of the ANME-2a/b clade. We propose that natural organic matter serving as electron acceptor drives anaerobic methanotrophy in brackish sediments.

Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Amsterdam urban canals contain novel niches for methane-cycling microorganisms.

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol gDW -1  d-1 that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.

Insect Gut Isolate Pseudomonas sp. Strain Nvir Degrades the Toxic Plant Metabolite Nitropropionic Acid.

Nitropropionic acid (NPA) is a widely distributed naturally occurring nitroaliphatic toxin produced by leguminous plants and fungi. The Southern green shield bug feeds on leguminous plants and shows no symptoms of intoxication. Likewise, its gut-associated microorganisms are subjected to high levels of this toxic compound. In this study, we isolated a bacterium from this insect's gut system, classified as Pseudomonas sp. strain Nvir, that was highly resistant to NPA and was fully degrading it to inorganic nitrogen compounds and carbon dioxide. In order to understand the metabolic fate of NPA, we traced the fate of all atoms of the NPA molecule using isotope tracing experiments with [15N]NPA and [1-13C]NPA, in addition to experiments with uniformly 13C-labeled biomass that was used to follow the incorporation of 12C atoms from [U-12C]NPA into tricarboxylic acid cycle intermediates. With the help of genomics and transcriptomics, we uncovered the isolate's NPA degradation pathway, which involves a putative propionate-3-nitronate monooxygenase responsible for the first step of NPA degradation. The discovered protein shares only 32% sequence identity with previously described propionate-3-nitronate monooxygenases. Finally, we advocate that NPA-degrading bacteria might find application in biotechnology, and their unique enzymes might be used in biosynthesis, bioremediation, and in dealing with postharvest NPA contamination in economically important products. IMPORTANCE Plants have evolved sophisticated chemical defense mechanisms, such as the production of plant toxins in order to deter herbivores. One example of such a plant toxin is nitropropionic acid (NPA), which is produced by leguminous plants and also by certain fungi. In this project, we have isolated a bacterium from the intestinal tract of a pest insect, the Southern green shield bug, that is able to degrade NPA. Through a multiomics approach, we identified the respective metabolic pathway and determined the metabolic fate of all atoms of the NPA molecule. In addition, we provide a new genetic marker that can be used for genome mining toward NPA degradation. The discovery of degradation pathways of plant toxins by environmental bacteria opens new possibilities for pretreatment of contaminated food and feed sources and characterization of understudied enzymes allows their broad application in biotechnology.

The Polar Fox Lagoon in Siberia harbours a community of Bathyarchaeota possessing the potential for peptide fermentation and acetogenesis.

Archaea belonging to the phylum Bathyarchaeota are the predominant archaeal species in cold, anoxic marine sediments and additionally occur in a variety of habitats, both natural and man-made. Metagenomic and single-cell sequencing studies suggest that Bathyarchaeota may have a significant impact on the emissions of greenhouse gases into the atmosphere, either through direct production of methane or through the degradation of complex organic matter that can subsequently be converted into methane. This is especially relevant in permafrost regions where climate change leads to thawing of permafrost, making high amounts of stored carbon bioavailable. Here we present the analysis of nineteen draft genomes recovered from a sediment core metagenome of the Polar Fox Lagoon, a thermokarst lake located on the Bykovsky Peninsula in Siberia, Russia, which is connected to the brackish Tiksi Bay. We show that the Bathyarchaeota in this lake are predominantly peptide degraders, producing reduced ferredoxin from the fermentation of peptides, while degradation pathways for plant-derived polymers were found to be incomplete. Several genomes encoded the potential for acetogenesis through the Wood-Ljungdahl pathway, but methanogenesis was determined to be unlikely due to the lack of genes encoding the key enzyme in methanogenesis, methyl-CoM reductase. Many genomes lacked a clear pathway for recycling reduced ferredoxin. Hydrogen metabolism was also hardly found: one type 4e [NiFe] hydrogenase was annotated in a single MAG and no [FeFe] hydrogenases were detected. Little evidence was found for syntrophy through formate or direct interspecies electron transfer, leaving a significant gap in our understanding of the metabolism of these organisms.

A novel methoxydotrophic metabolism discovered in the hyperthermophilic archaeon Archaeoglobus fulgidus.

Methoxylated aromatic compounds (MACs) are important components of lignin found in significant amounts in the subsurface. Recently, the methanogenic archaeon Methermicoccus shengliensis was shown to be able to use a variety of MACs during methoxydotrophic growth. After a molecular survey, we found that the hyperthermophilic non-methanogenic archaeon Archaeoglobus fulgidus also encodes genes for a bacterial-like demethoxylation system. In this study, we performed growth and metabolite analysis, and used transcriptomics to investigate the response of A. fulgidus during growth on MACs in comparison to growth on lactate. We observed that A. fulgidus converts MACs to their hydroxylated derivatives with CO2 as the main product and sulfate as electron acceptor. Furthermore, we could show that MACs improve the growth of A. fulgidus in the presence of organic substrates such as lactate. We also found evidence that other archaea such as Bathyarchaeota, Lokiarchaeota, Verstraetearchaeota, Korarchaeota, Helarchaeota and Nezhaarchaeota encode a demethoxylation system. In summary, we here describe the first non-methanogenic archaeon with the ability to grow on MACs indicating that methoxydotrophic archaea might play a so far underestimated role in the global carbon cycle.

Do initial concentration and activated sludge seasonality affect pharmaceutical biotransformation rate constants?

Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC-MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. KEY POINTS: • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change.

Several ways one goal-methanogenesis from unconventional substrates.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways. KEY POINTS: • Newly discovered methanogenic substrates and pathways are reviewed for the first time. • The review provides an in-depth analysis of unusual methanogenesis pathways. • The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

High-Level Abundances of Methanobacteriales and Syntrophobacterales May Help To Prevent Corrosion of Metal Sheet Piles.

Iron sheet piles are widely used in flood protection, dike construction, and river bank reinforcement. Their corrosion leads to gradual deterioration and often makes replacement necessary. Natural deposit layers on these sheet piles can prevent degradation and significantly increase their life span. However, little is known about the mechanisms of natural protective layer formation. Here, we studied the microbially diverse populations of corrosion-protective deposit layers on iron sheet piles at the Gouderak pumping station in Zuid-Holland, the Netherlands. Deposit layers, surrounding sediment and top sediment samples were analyzed for soil physicochemical parameters, microbially diverse populations, and metabolic potential. Methanogens appeared to be enriched 18-fold in the deposit layers. After sequencing, metagenome assembly and binning, we obtained four nearly complete draft genomes of microorganisms (Methanobacteriales, two Coriobacteriales, and Syntrophobacterales) that were highly enriched in the deposit layers, strongly indicating a potential role in corrosion protection. Coriobacteriales and Syntrophobacterales could be part of a microbial food web degrading organic matter to supply methanogenic substrates. Methane-producing Methanobacteriales could metabolize iron, which may initially lead to mild corrosion but potentially stimulates the formation of a carbonate-rich protective deposit layer in the long term. In addition, Methanobacteriales and Coriobacteriales have the potential to interact with metal surfaces via direct interspecies or extracellular electron transfer. In conclusion, our study provides valuable insights into microbial populations involved in iron corrosion protection and potentially enables the development of novel strategies for in situ screening of iron sheet piles in order to reduce risks and develop more sustainable replacement practices.IMPORTANCE Iron sheet piles are widely used to reinforce dikes and river banks. Damage due to iron corrosion poses a significant safety risk and has significant economic impact. Different groups of microorganisms are known to either stimulate or inhibit the corrosion process. Recently, natural corrosion-protective deposit layers were found on sheet piles. Analyses of the microbial composition indicated a potential role for methane-producing archaea. However, the full metabolic potential of the microbial communities within these protective layers has not been determined. The significance of this work lies in the reconstruction of the microbial food web of natural corrosion-protective layers isolated from noncorroding metal sheet piles. With this work, we provide insights into the microbiological mechanisms that potentially promote corrosion protection in freshwater ecosystems. Our findings could support the development of screening protocols to assess the integrity of iron sheet piles to decide whether replacement is required.

Increases in temperature and nutrient availability positively affect methane-cycling microorganisms in Arctic thermokarst lake sediments.

Arctic permafrost soils store large amounts of organic matter that is sensitive to temperature increases and subsequent microbial degradation to methane (CH 4 ) and carbon dioxide (CO 2 ). Here, we studied methanogenic and methanotrophic activity and community composition in thermokarst lake sediments from Utqiag˙vik (formerly Barrow), Alaska. This experiment was carried out under in situ temperature conditions (4°C) and the IPCC 2013 Arctic climate change scenario (10°C) after addition of methanogenic and methanotrophic substrates for nearly a year. Trimethylamine (TMA) amendment with warming showed highest maximum CH 4 production rates, being 30% higher at 10°C than at 4°C. Maximum methanotrophic rates increased by up to 57% at 10°C compared to 4°C. 16S rRNA gene sequencing indicated high relative abundance of Methanosarcinaceae in TMA amended incubations, and for methanotrophic incubations Methylococcaeae were highly enriched. Anaerobic methanotrophic activity with nitrite or nitrate as electron acceptor was not detected. This study indicates that the methane cycling microbial community can adapt to temperature increases and that their activity is highly dependent on substrate availability.

Functional Profiling and Crystal Structures of Isothiocyanate Hydrolases Found in Gut-Associated and Plant-Pathogenic Bacteria.

Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolases (ITCases), a novel family within the metallo-ß-lactamase (MBL)-fold superfamily of proteins. saxA genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases revealed similar activities on six selected ITCs, suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCases include a hydrophobic active site with two Zn2+ ions coordinating water/hydroxide and a flexible cap that is implicated in substrate recognition and covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-ß-lactamases.IMPORTANCE This study explores a newly discovered protein in the ß-lactamase superfamily, namely, SaxA, or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals in vitro and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Cre-inducible site-specific recombination in zebrafish oligodendrocytes.

BACKGROUND: The conditional Cre/lox system has recently emerged as a valuable tool for studies on both embryonic and adult Zebrafish. Temporal control and site-specific recombination are achieved by using the ligand-inducible CreERT2 and administration of the drug tamoxifen (TAM) or its active metabolite, 4-Hydroxytamoxifen (4-OHT). RESULTS: Here we report the generation of a transgenic Zebrafish line, which expresses an mCherry-tagged variant of CreERT2 under the control of the myelin basic protein a (mbpa) promoter. Our analysis shows that larval and adult expression of the transgene recapitulates the endogenous mbpa expression pattern in oligodendrocytes. Furthermore, combination with a Cre-dependent EGFP reporter results in EGFP-expressing oligodendrocytes in the spinal cord, brain, and optic nerve in TAM- or 4-OHT-treated larvae and 4-month-old fish, but not in untreated controls. CONCLUSIONS: The transgenic Zebrafish line Tg(mbpa:mCherry-T2A-CreERT2 ) elicits CreERT2 expression specifically in myelinating glia cells. Cre-inducible targeted recombination of genes in oligodendrocytes will be useful to elucidate cellular and molecular mechanisms of myelination in vivo during development (myelination) and regeneration (remyelination) after injury to the central nervous system (CNS). It will also allow targeted expression and overexpression of genes of interest (transgenes) in oligodendrocytes at defined developmental and adult stages. Developmental Dynamics 246:41-49, 2017. © 2016 Wiley Periodicals, Inc.

Mimicking microbial interactions under nitrate-reducing conditions in an anoxic bioreactor: enrichment of novel Nitrospirae bacteria distantly related to Thermodesulfovibrio.

Microorganisms are main drivers of the sulfur, nitrogen and carbon biogeochemical cycles. These elemental cycles are interconnected by the activity of different guilds in sediments or wastewater treatment systems. Here, we investigated a nitrate-reducing microbial community in a laboratory-scale bioreactor model that closely mimicked estuary or brackish sediment conditions. The bioreactor simultaneously consumed sulfide, methane and ammonium at the expense of nitrate. Ammonium oxidation occurred solely by the activity of anammox bacteria identified as Candidatus Scalindua brodae and Ca. Kuenenia stuttgartiensis. Fifty-three percent of methane oxidation was catalyzed by archaea affiliated to Ca. Methanoperedens and 47% by Ca. Methylomirabilis bacteria. Sulfide oxidation was mainly shared between two proteobacterial groups. Interestingly, competition for nitrate did not lead to exclusion of one particular group. Metagenomic analysis showed that the most abundant taxonomic group was distantly related to Thermodesulfovibrio sp. (87-89% 16S rRNA gene identity, 52-54% average amino acid identity), representing a new family within the Nitrospirae phylum. A high quality draft genome of the new species was recovered, and analysis showed high metabolic versatility. Related microbial groups are found in diverse environments with sulfur, nitrogen and methane cycling, indicating that these novel Nitrospirae bacteria might contribute to biogeochemical cycling in natural habitats.

Reverse Methanogenesis and Respiration in Methanotrophic Archaea.

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

Plasmids from the gut microbiome of cabbage root fly larvae encode SaxA that catalyses the conversion of the plant toxin 2-phenylethyl isothiocyanate.

Cabbage root fly larvae (Delia radicum) cause severe crop losses (≥ 50%) of rapeseed/ canola and cabbages used in the food and biofuel industries. These losses occur despite the fact that cabbages produce insecticidal toxins such as isothiocyanates. Here we describe the cabbage root fly larval gut microbiome as a source of isothiocyanate degrading enzymes. We sequenced the microbial gut community of the larvae and analysed phylogenetic markers and functional genes. We combined this with the isolation of several microbial strains representing the phylogenetic distribution of the metagenome. Eleven of those isolates were highly resistant towards 2-phenylethyl isothiocyanate, a subset also metabolized 2-phenylethyl isothiocyanate. Several plasmids appeared to be shared between those isolates that metabolized the toxin. One of the plasmids harboured a saxA gene that upon transformation gave resistance and enabled the degradation of 2-phenylethyl isothiocyanate in Escherichia coli. Taken together, the results showed that the cabbage root fly larval gut microbiome is capable of isothiocyanate degradation, a characteristic that has not been observed before, and may help us understand and design new pest control strategies.