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The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatusbafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Mechanisms controlling CO2 and CH4 production in wetlands are central to understanding carbon cycling and greenhouse gas exchange. However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO2 and CH4 dynamics.

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins-Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

The Gram-negative bacterium Vibrio cholerae is responsible for the severe diarrheal pandemic disease cholera, representing a major global public health concern. This pathogen transitions from aquatic reservoirs into epidemics in human populations, and has evolved numerous mechanisms to sense this transition in order to appropriately regulate its gene expression for infection. At the intersection of pathogen and host in the gastrointestinal tract lies the community of native gut microbes, the gut microbiome. It is increasingly clear that the diversity of species and biochemical activities within the gut microbiome represents a driver of infection outcome, through their ability to manipulate the signals used by V. cholerae to regulate virulence and fitness in vivo. A better mechanistic understanding of how commensal microbial action interacts with V. cholerae pathogenesis may lead to novel prophylactic and therapeutic interventions for cholera. Here, we review a subset of this burgeoning field of research.

Mercury (Hg) methylation is a microbially mediated process that converts inorganic Hg into bioaccumulative, neurotoxic methylmercury (MeHg). The metabolic activity of methylating organisms is highly dependent on biogeochemical conditions, which subsequently influences MeHg production. However, our understanding of the ecophysiology of methylators in natural ecosystems is still limited. Here, we identified potential locations of MeHg production in the anoxic, sulfidic hypolimnion of a freshwater lake. At these sites, we used shotgun metagenomics to characterize microorganisms with the Hg-methylation gene hgcA. Putative methylators were dominated by hgcA sequences divergent from those in well-studied, confirmed methylators. Using genome-resolved metagenomics, we identified organisms with hgcA (hgcA+) within the Bacteroidetes and the recently described Kiritimatiellaeota phyla. We identified hgcA+ genomes derived from sulfate-reducing bacteria, but these accounted for only 22% of hgcA+ genome coverage. The most abundant hgcA+ genomes were from fermenters, accounting for over half of the hgcA gene coverage. Many of these organisms also mediate hydrolysis of polysaccharides, likely from cyanobacterial blooms. This work highlights the distribution of the Hg-methylation genes across microbial metabolic guilds and indicate that primary degradation of polysaccharides and fermentation may play an important but unrecognized role in MeHg production in the anoxic hypolimnion of freshwater lakes.

BackgroundAnaerobic ammonium oxidation (anammox) is a biological process employed to remove reactive nitrogen from wastewater. While a substantial body of literature describes the performance of anammox bioreactors under various operational conditions and perturbations, few studies have resolved the metabolic roles of their core microbial community members.ResultsHere, we used metagenomics to study the microbial community of a laboratory-scale anammox bioreactor from inoculation, through a performance destabilization event, to robust steady-state performance. Metabolic analyses revealed that nutrient acquisition from the environment is selected for in the anammox community. Dissimilatory nitrate reduction to ammonium (DNRA) was the primary nitrogen removal pathway that competed with anammox. Increased replication of bacteria capable of DNRA led to the out-competition of anammox bacteria, and the loss of the bioreactor's nitrogen removal capacity. These bacteria were highly associated with the anammox bacterium and considered part of the core microbial community.ConclusionsOur findings highlight the importance of metabolic interdependencies related to nitrogen- and carbon-cycling within anammox bioreactors and the potentially detrimental effects of bacteria that are otherwise considered core microbial community members.

The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.

Anaerobic bacteria are a promising new source for natural product discovery. Examination of extracts from the obligate anaerobe Clostridium roseum led to the discovery of a new family of natural products, the clostyrylpyrones. The polyketide synthase-based biosynthetic mechanism of clostyrylpyrones is further proposed on the basis of bioinformatic, gene knockout, biochemical analysis, and heterologous expression studies.

The availability of labile carbon (C) compounds in Arctic wetland soils is expected to increase due to thawing permafrost and increased fermentation as a result of decomposition of organic matter with warming. How microbial communities respond to this change will affect the balance of CO2 and CH4 emitted during anaerobic organic matter decomposition, and ultimately the net radiative forcing of greenhouse gas emissions from these soils. While soil water content limits aerobic respiration, the factors controlling methanogenesis and anaerobic respiration are poorly defined in suboxic Arctic soils. We conducted incubation experiments on two tundra soils from field sites on the Seward Peninsula, Alaska, with contrasting pH and geochemistry to determine the pathways of anaerobic microbial respiration and changes with increasing substrate availability upon warming. In incubation of soils from the circumneutral Teller site, the ratio of CO2 to CH4 dropped from 10 to

Abstract Aim Cycling processes in wetlands are highly dynamic and involve complex interactions between hydrological processes, biogeochemical transformations, and microbial communities. This review attempts to assess the interactions between elements within biogeochemical cycles and the possible routes in which organic matter is processed in waterlogged soils. Methods The input and cycling of organic matter in flooded soils were approached in this review. We used a non-systematic literature survey to indicate the possible biogeochemical routes of organic matter processing in waterlogged soils. Results We explore hydrological processes, oxygen availability, biogeochemical routes of the organic matter process, and the inputs and exports of organic matter in flooded soils from wetlands. Conclusions The anaerobic degradation of organic resources predominantly occurs within submerged soils. Under conditions of maintenance of natural rates of primary production and allochthonous detritus input, storing organic detritus in flooded soils prevails over mineralization. The importance of hydrology for the export of organic carbon is evident. In wetlands, the export of organic matter is predominantly associated with dissolved organic matter and methane production.

Once thought to be a waste product of oxygen limited (anaerobic) metabolism, lactate is now known to form continuously under fully oxygenated (aerobic) conditions. Lactate shuttling between producer (driver) and consumer cells fulfills at least 3 purposes; lactate is: (1) a major energy source, (2) the major gluconeogenic precursor, and (3) a signaling molecule. The Lactate Shuttle theory is applicable to diverse fields such as sports nutrition and hydration, resuscitation from acidosis and Dengue, treatment of traumatic brain injury, maintenance of glycemia, reduction of inflammation, cardiac support in heart failure and following a myocardial infarction, and to improve cognition. Yet, dysregulated lactate shuttling disrupts metabolic flexibility, and worse, supports oncogenesis. Lactate production in cancer (the Warburg effect) is involved in all main sequela for carcinogenesis: angiogenesis, immune escape, cell migration, metastasis, and self-sufficient metabolism. The history of the tortuous path of discovery in lactate metabolism and shuttling was discussed in the 2019 American College of Sports Medicine Joseph B. Wolffe Lecture in Orlando, FL.

Tedizolid's anaerobic activity is unappreciated. In this study, it was active against all 332 anaerobic isolates tested at ≤2 μg/ml except Bilophila wadsworthia and was more active than linezolid against Bacteroides fragilis group species (MIC90, 1 μg/ml versus 2 to 4 μg/ml). Tedizolid was active against Gram-positive anaerobes (MIC90 for clostridia, 0.25 to 1 μg/ml; MIC90 for anaerobic cocci, ≤0.06 to 0.25 μg/ml). Our data coupled with clinical reports indicate that clinicians should consider its use in mixed infections where Staphylococcus aureus and anaerobes are involved.

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H2. Methanosarcina thermophila, the predominant methanogen prior to H2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H2, which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches. Video abstract.

Isoprenoids are an essential and diverse class of molecules, present in all forms of life, that are synthesized from an essential common precursor derived from either the mevalonate pathway or the nonmevalonate pathway. Most bacteria have one pathway or the other, but the Gram-positive, facultative intracellular pathogen Listeria monocytogenes is unusual because it carries all the genes for both pathways. While the mevalonate pathway has previously been reported to be essential, here we demonstrate that the nonmevalonate pathway can support growth of strains 10403S and EGD-e, but only anaerobically. L. monocytogenes lacking the gene hmgR, encoding the rate-limiting enzyme of the mevalonate pathway, had a doubling time of 4 h under anaerobic conditions, in contrast to the 45 min doubling time of the wild type. In contrast, deleting hmgR in two clinical isolates resulted in mutants that grew significantly faster, doubling in approximately 2 h anaerobically, although they still failed to grow under aerobic conditions without mevalonate. The difference in anaerobic growth rate was traced to three amino acid changes in the nonmevalonate pathway enzyme GcpE, and these changes were sufficient to increase the growth rate of 10403S to the rate observed in the clinical isolates. Despite an increased growth rate, virulence was still dependent on the mevalonate pathway in 10403S strains expressing the more active GcpE allele.

Oxygenic photosynthesis supplies organic carbon to the modern biosphere, but it is uncertain when this metabolism originated. It has previously been proposed1,2 that photosynthetic reaction centres capable of splitting water arose by about 3 billion years ago on the basis of the inferred presence of manganese oxides in Archaean sedimentary rocks. However, this assumes that manganese oxides can be produced only in the presence of molecular oxygen3, reactive oxygen species4,5 or by high-potential photosynthetic reaction centres6,7. Here we show that communities of anoxygenic photosynthetic microorganisms biomineralize manganese oxides in the absence of molecular oxygen and high-potential photosynthetic reaction centres. Microbial oxidation of Mn(II) under strictly anaerobic conditions during the Archaean eon would have produced geochemical signals identical to those used to date the evolution of oxygenic photosynthesis before the Great Oxidation Event1,2. This light-dependent process may also produce manganese oxides in the photic zones of modern anoxic water bodies and sediments.

Biogas is a renewable energy source composed of methane, carbon dioxide, and other trace compounds produced from anaerobic digestion of organic matter. A variety of feedstocks can be combined with different digestion techniques that each yields biogas with different trace compositions. California is expanding biogas production systems to help meet greenhouse gas reduction goals. Here, we report the composition of six California biogas streams from three different feedstocks (dairy manure, food waste, and municipal solid waste). The chemical and biological composition of raw biogas is reported, and the toxicity of combusted biogas is tested under fresh and photochemically aged conditions. Results show that municipal waste biogas contained elevated levels of chemicals associated with volatile chemical products such as aromatic hydrocarbons, siloxanes, and certain halogenated hydrocarbons. Food waste biogas contained elevated levels of sulfur-containing compounds including hydrogen sulfide, mercaptans, and sulfur dioxide. Biogas produced from dairy manure generally had lower concentrations of trace chemicals, but the combustion products had slightly higher toxicity response compared to the other feedstocks. Atmospheric aging performed in a photochemical smog chamber did not strongly change the toxicity (oxidative capacity or mutagenicity) of biogas combustion exhaust.

Locomotion is a defining characteristic that can dictate many aspects of an organism's life history in the pursuit of maximizing fitness, including escaping predators, capturing prey, and transitioning between habitats. Exhaustive exercise can have negative consequences for both short-term and long-term energetics and life history trade-offs, influencing fish survival and reproduction. Studies of swimming performance and exhaustive exercise in fish are often conducted on individual species, but few multispecies analyses exist and even fewer in field settings. In fish, swimming performance and exercise have historically been studied in the laboratory using swim tunnels, but an increasing body of work in recreational fisheries science provides a novel way to examine swimming capacity and exhaustion. Using fight time, the time it takes for a hooked fish to be landed on rod and reel fishing gear, as an opportunistic proxy for fish exhaustion, a multispecies meta-analysis of data from studies on recreational fisheries was conducted to elucidate the factors that most influence capacity for exhaustive exercise. Data from 39 species of freshwater and marine fish were aggregated, and negative binomial mixed effects models as well as phylogenetic least squares regression were used to identify the factors that most influenced exhaustive exercise in the field. Fish total length, aspect ratio of the caudal fin, and body form were significant factors in explaining the capacity for exhaustive exercise. Large migratory fish with high aspect ratios were able to fight, and therefore exercise, the longest. These results illustrate that body form and physiology are both deeply intertwined to inform function across fish species and point to angling fight time as a useful approximation of fish swimming capabilities that can be further developed for understanding the limits of fish exercise physiology. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the effects of different proportions of cactus pear mucilage as a moistening additive on chemical composition, ammonia nitrogen, volatile fatty acids, microbial dynamics, fermentation losses, pH, and aerobic stability of corn grains in the form of silage throughout storage periods. The study was carried out in a completely randomized design with a factorial arrangement (5×2), with four replications. The factors corresponded to five forms of moistening for corn grain silage, 0, 5, 10, 20, and 40% of cactus pear mucilage (MA) associated or not with urea (U) in the level of 1.5% of DM. The lowest DM content was found in the silage moistened with 40% of cactus pear mucilage, and the greatest CP content was found in the same treatment but when urea was added. Lower contents of propionic and butyric acids were observed in the silages moistened with 5 and 10% of cactus pear without and with the addition of urea. Dry matter recovery was higher in the treatments with water and 10% of cactus pear. The use of urea affected the LAB population only at 15 d of silo opening, providing a bigger population (6.58 logs CFU g -1 ) when compared to silages without urea (6.27 logs CFU g -1 ). The lower concentrations of propionic and butyric acids observed in the silage moistened with 5 and 10% of cactus pear mucilage, regardless of the addition of urea. However, it is recommended to use 10% of cactus pear mucilage without the addition of urea, as it provides better indicators of good-quality silage. [ABSTRACT FROM AUTHOR]

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The study of biodeterioration is an important issue to allow the best conservation and prevent the decay of cultural heritage and artworks. In Naples (Italy), a particular museum (Museodivino) preserves the miniature artworks representing Dante’s Divine Comedy and Nativity scenes, executed with organic-based materials in walnut and clay shells. Since they showed putative signs of biodeterioration, the first aim of this study was to verify the presence of microbial colonization. A culture-dependent approach and molecular biology allowed us to isolate and identify the sole fungal strain Aspergillus NCCD (Nativity and Dante’s Divine Comedy) belonging to the A. sydowii sub-clade. Based on this result, a sustainable and eco-friendly approach was applied to find a method to preserve the miniature artwork by contrasting the growth of the strain NCCD. Several essential oils used as a natural biocide were tested against Aspergillus strain NCCD belonging to the A. sydowii subclade to determine their potential antimicrobial activity. Results revealed that basil, cloves, fennel, and thyme essential oils exerted antifungal activity, although their effect depended also on the concentration used. Moreover, anoxic treatment and the control of the relative humidity were used in the presence of thyme, in vitro, and in vivo assays to define the impact on fungal growth. No fungal development was detected in vivo in the shells treated with thyme essential oil at high relative humidity after 60 days of incubation at 28 °C. These results highlighted that although relative humidity was the major factor affecting the development of the strain Aspergillus NDDC, the application of thyme in an anaerobic environment is essential in contrasting the fungal growth. Identifying the biodeterioration agent allowed us to plan an eco-friendly, non-destructive approach to be successfully used to guarantee the conditions suitable for conserving miniature artwork.

Anaerobic digestion is one of the most successful waste management strategies worldwide, wherein microorganisms play an essential role in reducing organic pollutants and producing renewable energy. However, variations of microbial community in full-scale anaerobic digesters, particularly functional groups relevant to biogas production, remain elusive. Here, we examined microbial community in a year-long monthly time series of 3 full-scale anaerobic digesters. We observed substantial diversification in community composition, with only a few abundant OTUs (e.g. Clostridiales, Anaerolineaceae and Methanosaeta) persistently present across different samples. Similarly, there were high variations in relative abundance of methanogenic archaea and methanogenic genes, which were positively correlated (r2 = 0.530, P

The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H2 metabolism were central metabolic features. The ability to use O2 as a terminal electron acceptor must have been subsequently acquired by these lineages.

An overview of modern methods used in the preparation and characterization of molybdenum-containing enzymes is presented, with an emphasis on those methods that have been developed over the past decade to address specific difficulties frequently encountered in studies of these enzymes.

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.

The U.S. places approximately 53% of its total municipal solid waste (MSW) in landfills, but state and local governments across the country are now setting ambitious environmental and waste diversion policies requiring, among other things, diversion and utilization of organics. Municipalities across the U.S. are employing anaerobic digestion (AD) as part of their strategy to divert organic MSW from landfills, produce biogas, and yield other beneficial coproducts such as compost and fertilizer. However, AD faces many technical, regulatory, and economic barriers to greater deployment, including upstream waste contamination, local odor and air pollution concerns, lengthy siting and permitting processes, and requirements and sizable costs for interconnecting to the electric grid. We identify a combination of scientific, operational, and policy advancements that are needed to address these barriers.

Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to optimize hydrolytic efficiency. Our analysis of anaerobic fungal transcriptomes reveals hundreds of cis-natural antisense transcripts (cis-NATs), which we hypothesize play an integral role in this regulation. Through integrated genomic and transcriptomic sequencing on a range of catabolic substrates, we validate these NATs in three species (Anaeromyces robustus, Neocallimasix californiae, and Piromyces finnis), and analyze their expression patterns and prevalence to gain insight into their function. NAT function was diverse and conserved across the three fungal genomes studied, with 10% of all metabolic process NATs associated with lignocellulose hydrolysis. Despite these similarities, however, only eleven gene targets were conserved orthologs. Several NATs were dynamically regulated by lignocellulosic substrates while their gene targets were unregulated. This observation is consistent with a hypothesized, but untested, regulatory mechanism where selected genes are exclusively regulated at the transcriptional/post-transcriptional level by NATs. However, only genes with high NAT relative expression levels displayed this phenomenon, suggesting a selection mechanism that favors larger dynamic ranges for more precise control of gene expression. In addition to this mode, we observed two other possible regulatory fates: canonical transcriptional regulation with no NAT response, and positive co-regulation of target mRNA and cognate NAT, which we hypothesize is a fine-tuning strategy to locally negate control outputs from global regulators. Our work reveals the complex contributions of antisense RNA to the catabolic response in anaerobic fungi, highlighting its importance in understanding lignocellulolytic activity for bioenergy applications. More importantly, the relative expression of NAT to target may form a critical determinant of transcriptional vs post-transcriptional (NAT) control of gene expression in primitive anaerobic fungi.

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

Anaerobic ammonium oxidation (anammox) contributes substantially to ocean nitrogen loss, particularly in anoxic marine zones (AMZs). Ammonium is scarce in AMZs, raising the hypothesis that organic nitrogen compounds may be ammonium sources for anammox. Biochemical measurements suggest that the organic compounds urea and cyanate can support anammox in AMZs. However, it is unclear if anammox bacteria degrade these compounds to ammonium themselves, or rely on other organisms for this process. Genes for urea degradation have not been found in anammox bacteria, and genomic evidence for cyanate use for anammox is limited to a cyanase gene recovered from the sediment bacterium Candidatus Scalindua profunda. Here, analysis of Ca. Scalindua single amplified genomes from the Eastern Tropical North Pacific AMZ revealed genes for urea degradation and transport, as well as for cyanate degradation. Urease and cyanase genes were transcribed, along with anammox genes, in the AMZ core where anammox rates peaked. Homologs of these genes were also detected in meta-omic datasets from major AMZs in the Eastern Tropical South Pacific and Arabian Sea. These results suggest that anammox bacteria from different ocean regions can directly access organic nitrogen substrates. Future studies should assess if and under what environmental conditions these substrates contribute to the ammonium budget for anammox.

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-l-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.

Candidate phyla radiation (CPR) bacteria and DPANN (an acronym of the names of the first included phyla) archaea are massive radiations of organisms that are widely distributed across Earth's environments, yet we know little about them. Initial indications are that they are consistently distinct from essentially all other bacteria and archaea owing to their small cell and genome sizes, limited metabolic capacities and often episymbiotic associations with other bacteria and archaea. In this Analysis, we investigate their biology and variations in metabolic capacities by analysis of approximately 1,000 genomes reconstructed from several metagenomics-based studies. We find that they are not monolithic in terms of metabolism but rather harbour a diversity of capacities consistent with a range of lifestyles and degrees of dependence on other organisms. Notably, however, certain CPR and DPANN groups seem to have exceedingly minimal biosynthetic capacities, whereas others could potentially be free living. Understanding of these microorganisms is important from the perspective of evolutionary studies and because their interactions with other organisms are likely to shape natural microbiome function.

BACKGROUND: The present study aimed to determine the optimal conditions for making wines by the carbonic maceration (CM) method. In this test, we attempted to identify the causes of the higher volatile acidity of some wines vinified by this method. Accordingly, we measured the development and speed of intracellular fermentation inside the whole grapes under different vinification conditions. RESULTS: An active fermentation of the must in the tanks produced by inoculation with active dry yeasts was more efficient for the process than the addition of exogenous carbon dioxide (CO2). In addition, in CM vinification, the moment of devatting had a great influence on the content of acetaldehyde and acetic acid in the whole grapes. CONCLUSION: Yeast inoculation in the tanks and control of the devatting time are technological tools that could play an important role in the characteristics of the wines produced by carbonic maceration, especially with respect to acetic acid content. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract Background Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. Results We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5′-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and d-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. Conclusions This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

Gut bacterial dysbiosis has been linked to several gastrointestinal diseases, including deadly colorectal cancer (CRC), a leading cause of mortality in cancer patients. However, perturbation in gut bacteriome during colon cancer (CC, devoid of colorectal malignancy) remains poorly explored. Here, 16S rRNA gene amplicon sequencing was carried out for fecal DNA samples targeted to hypervariable V3–V4 region by employing MiSeq platform to explore the gut bacterial community shift in CC patients. While alpha diversity indices predicted high species richness and diversity, beta diversity showed marked gut bacterial compositional dissimilarity in CC versus healthy controls (HC, n = 10 each). We observed a significant (p < 0.05, Wilcoxon Rank-Sum test) emergence of low-abundant anaerobic taxa, including Parvimonas and Peptostreptococcus , in addition to Subdoligranulum , Coprococcus , Holdemanella, Solobacterium , Bilophila , Blautia, Dorea , Moryella and several unidentified taxa, mainly affiliated to Firmicutes , in CC patients. In addition, we also traced the emergence of putative probiotic taxon Slackia , belonging to Actinomycetota , in CC patients. The emergence of anaerobic Firmicutes in CC is accompanied by a significant (p < 0.05) decline in the Klebsiella , as determined through linear discriminant analysis effect size (LEfSe) and heat tree analyses. Shifts in core microbiome and variation in network correlation were also witnessed. Taken together, this study highlighted a significant and consistent emergence of rare anaerobic Firmicutes suggesting possible anaerobiosis driving gut microbial community shift, which could be exploited in designing diagnostic and therapeutic tools targeted to CC. • Emergence of low-abundant anaerobic Firmicutes , including Parvimonas , Peptostreptococcus and Moryella , in colon cancer. • Probiotic taxon Slackia emerged in colon cancer suggesting potential implications for therapeutic interventions. • Klebsiella significantly declined in the colon cancer gut microbiome indicating a shift towards anaerobiosis [ABSTRACT FROM AUTHOR]

The aim of the present study was to validate methods of stool sample conservation for the egg hatch test (EHT). This study involved the use of a bovine naturally infected predominantly by Cooperia spp. and one equine naturally infected predominantly by cyathostomins characterized as susceptible to benzimidazoles in the EHT. Fecal samples were submitted to three treatments: aerobic methods (anaerobic storage in plastic bottles, anaerobic storage in vacuum-sealed bags or aerobic storage in plastic bags), under two temperature conditions (room temperature and refrigeration) analyzed at four different assessment times (48, 72, 96 and 120 h). As the standard test, an assay was also performed within 3 h. The tests were performed in triplicate for each drug concentration and with three experimental repetitions at one-week intervals. Two criteria were used for the storage methods: hatchability in the negative control group and sensitivity of the eggs to thiabendazole, comparing the EC50 and 95% confidence interval for each treatment to those of the standard test and the other repetitions. Bovine samples can be stored for up to 96 h and refrigerated vacuum storage can be used, ensuring hatchability of the negative control and sensitivity of the eggs to thiabendazole. For equine samples, no forms of storage were indicated due to the variation among the repetitions and the reduction in the sensitivity of the eggs to thiabendazole, which could result in a false positive detection of resistance. [Display omitted] • Bovine feces can be stored for up to 96 h, but the EHT must be performed within 3 h for equine feces samples. • Samples stored at room temperature have good hatchability but with reduced sensitivity to thiabendazole in the EHT. • Vacuum-sealed refrigerated samples ensure adequate hatchability. [ABSTRACT FROM AUTHOR]

Ocean temperatures will increase significantly over the next 100 years due to global climate change1. As temperatures increase beyond current ranges, it is unclear how adaptation will impact the distribution and ecological role of marine microorganisms2. To address this major unknown, we imposed a stressful high-temperature regime for 500 generations on a strain from the abundant marine Roseobacter clade. High-temperature-adapted isolates significantly improved their fitness but also increased biofilm formation at the air-liquid interface. Furthermore, this altered lifestyle was coupled with genomic changes linked to biofilm formation in individual isolates, and was also dominant in evolved populations. We hypothesize that the increasing biofilm formation was driven by lower oxygen availability at elevated temperature, and we observe a relative fitness increase at lower oxygen. The response is uniquely different from that of Escherichia coli adapted to high temperature3 (only 3% of mutated genes were shared in both studies). Thus, future increased temperatures could have a direct effect on organismal physiology and an indirect effect via a decrease in ocean oxygen solubility, leading to an alteration in microbial lifestyle.

Microbes of the phylum Aigarchaeota are widely distributed in geothermal environments, but their physiological and ecological roles are poorly understood. Here we analyze six Aigarchaeota metagenomic bins from two circumneutral hot springs in Tengchong, China, to reveal that they are either strict or facultative anaerobes, and most are chemolithotrophs that can perform sulfide oxidation. Applying comparative genomics to the Thaumarchaeota and Aigarchaeota, we find that they both originated from thermal habitats, sharing 1154 genes with their common ancestor. Horizontal gene transfer played a crucial role in shaping genetic diversity of Aigarchaeota and led to functional partitioning and ecological divergence among sympatric microbes, as several key functional innovations were endowed by Bacteria, including dissimilatory sulfite reduction and possibly carbon monoxide oxidation. Our study expands our knowledge of the possible ecological roles of the Aigarchaeota and clarifies their evolutionary relationship to their sister lineage Thaumarchaeota.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

Parasitic infections like amebiasis, trichomoniasis, and giardiasis are major health threats in tropical and subtropical regions of the world. Metronidazole (MTZ) is the current drug of choice for amebiasis, giardiasis, and trichomoniasis but it has several adverse effects and potential resistance is a concern. In order to develop alternative antimicrobials, a library of 1H-1,2,3-triazole-tethered metronidazole-isatin conjugates was synthesized using Huisgens azide-alkyne cycloaddition reaction and evaluated for their amebicidal, anti-trichomonal, and anti-giardial potential. Most of the synthesized conjugates exhibited activities against Trichomonas vaginalis, Tritrichomonas foetus, Entamoeba histolytica, and Giardia lamblia. While activities against T. vaginalis and T. foetus were comparable to that of the standard drug MTZ, better activities were observed against E. histolytica and G. lamblia. Conjugates 9d and 10a were found to be 2-3-folds more potent than MTZ against E. histolytica and 8-16-folds more potent than MTZ against G. lamblia. Further analysis of these compounds on fungi and bacteria did not show inhibitory activity, demonstrating their specific anti-protozoal properties.

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

Modern oncology aims at patient-specific therapy approaches, which triggered the development of biomedical imaging techniques to synergistically address tumor biology at the cellular and molecular level. PET/MR is a new hybrid modality that allows acquisition of high-resolution anatomic images and quantification of functional and metabolic information at the same time. Key steps of the Warburg effect-one of the hallmarks of tumors-can be measured non-invasively with this emerging technique. The aim of this study was to quantify and compare simultaneously imaged augmented glucose uptake and LDH activity in a subcutaneous breast cancer model in rats (MAT-B-III) and to study the effect of varying tumor cellularity on image-derived metabolic information. Methods: For this purpose, we established and validated a multimodal imaging workflow for a clinical PET/MR system including proton magnetic resonance (MR) imaging to acquire accurate morphologic information and diffusion-weighted imaging (DWI) to address tumor cellularity. Metabolic data were measured with dynamic [18F]FDG-PET and hyperpolarized (HP) 13C-pyruvate MR spectroscopic imaging (MRSI). We applied our workflow in a longitudinal study and analyzed the effect of growth dependent variations of cellular density on glycolytic parameters. Results: Tumors of similar cellularity with similar apparent diffusion coefficients (ADC) showed a significant positive correlation of FDG uptake and pyruvate-to-lactate exchange. Longitudinal DWI data indicated a decreasing tumor cellularity with tumor growth, while ADCs exhibited a significant inverse correlation with PET standard uptake values (SUV). Similar but not significant trends were observed with HP-13C-MRSI, but we found that partial volume effects and point spread function artifacts are major confounders for the quantification of 13C-data when the spatial resolution is limited and major blood vessels are close to the tumor. Nevertheless, analysis of longitudinal data with varying tumor cellularity further detected a positive correlation between quantitative PET and 13C-data. Conclusions: Our workflow allows the quantification of simultaneously acquired PET, MRSI and DWI data in rodents on a clinical PET/MR scanner. The correlations and findings suggest that a major portion of consumed glucose is metabolized by aerobic glycolysis in the investigated tumor model. Furthermore, we conclude that variations in cell density affect PET and 13C-data in a similar manner and correlations of longitudinal metabolic data appear to reflect both biochemical processes and tumor cellularity.

Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

China produces large amount of cotton stalk (CS) residues as agricultural biomass, which are incinerated on-site, causing air pollution. The high organic content of CS could be utilized for biogas production, but the direct digestion without pretreatment always leads to a low methane yield and biodegradability, due to the complicated structure of lignocellulose. In order to search best fitting pretreatment methods in effective anaerobic digestion (AD) of CS, effects of various pretreatments including KOH, NaOH, Ca(OH)2, alkali hydrogen peroxide (AHP), H2SO4, H3PO4 and steam explosion (SE) were studied. It was seen that all treatments resulted in varying methane yields. Among all the pretreatments, acid pretreatment is not suitable for AD of CS. The results showed that the highest cumulative methane yield (CMY) of 192.4 mL·gVS-1 was obtained after 3% AHP pretreatment of CS, and the methane yield improved by 254.3% than the untreated CS. Therefore, AHP treatment was proven to be an efficient pretreatment technique. XRD and FTIR analyses had shown that pretreated CS had favorable structural changes. This research is beneficial in developing environment friendly and cost-effective pretreatment technologies to utilize CS for methane production in future application.

Abstract Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.