The methane-oxidizing microbial communities of three maar lakes in tropical monsoon Asia.

Methane-oxidizing bacteria (MOB) is a group of planktonic microorganisms that use methane as their primary source of cellular energy. For tropical lakes in monsoon Asia, there is currently a knowledge gap on MOB community diversity and the factors influencing their abundance. Herewith, we present a preliminary assessment of the MOB communities in three maar lakes in tropical monsoon Asia using Catalyzed Reporter Deposition, Fluorescence In-Situ Hybridization (CARD-FISH), 16S rRNA amplicon sequencing, and pmoA gene sequencing. Correlation analysis between MOB abundances and lakes' physicochemical parameters following seasonal monsoon events were performed to explain observed spatial and temporal patterns in MOB diversity. The CARD-FISH analyses detected the three MOB types (I, II, and NC10) which aligned with the results from 16S rRNA amplicons and pmoA gene sequencing. Among community members based on 16S rRNA genes, Proteobacterial Type I MOB (e.g., Methylococcaceae and Methylomonadaceae), Proteobacterial Type II (Methylocystaceae), Verrucomicrobial (Methylacidiphilaceae), Methylomirabilota/NC10 (Methylomirabilaceae), and archaeal ANME-1a were found to be the dominant methane-oxidizers in three maar lakes. Analysis of microbial diversity and distribution revealed that the community compositions in Lake Yambo vary with the seasons and are more distinct during the stratified period. Temperature, DO, and pH were significantly and inversely linked with type I MOB and Methylomirabilota during stratification. Only MOB type I was influenced by monsoon changes. This research sought to establish a baseline for the diversity and ecology of planktonic MOB in tropical monsoon Asia to better comprehend their contribution to the CH4 cycle in tropical freshwater ecosystems.

Effect of oral tryptamines on the gut microbiome of rats-a preliminary study.

Background: Psilocybin and related tryptamines have come into the spotlight in recent years as potential therapeutics for depression. Research on the mechanisms of these effects has historically focused on the direct effects of these drugs on neural processes. However, in addition to such neural effects, alterations in peripheral physiology may also contribute to their therapeutic effects. In particular, substantial support exists for a gut microbiome-mediated pathway for the antidepressant efficacy of other drug classes, but no prior studies have determined the effects of tryptamines on microbiota. Methods: To address this gap, in this preliminary study, male Long Evans rats were treated with varying dosages of oral psilocybin (0.2 or 2 mg/kg), norbaeocystin (0.25 or 2.52 mg/kg), or vehicle and their fecal samples were collected 1 week and 3 weeks after exposure for microbiome analysis using integrated 16S ribosomal DNA sequencing to determine gut microbiome composition. Results: We found that although treatment with neither psilocybin nor norbaeocystin significantly affected overall microbiome diversity, it did cause significant dose- and time-dependent changes in bacterial abundance at the phylum level, including increases in Verrucomicrobia and Actinobacteria, and decreases in Proteobacteria. Conclusion and Implications: These preliminary findings support the idea that psilocybin and other tryptamines may act on the gut microbiome in a dose- and time-dependent manner, potentially identifying a novel peripheral mechanism for their antidepressant activity. The results from this preliminary study also suggest that norbaeocystin may warrant further investigation as a potential antidepressant, given the similarity of its effects to psilocybin.

Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods.

Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray-Curtis, P = 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%) Opitutae (Verrucomicrobia) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness.IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15-120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host-symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod-bacteria relationships and provides a foundation to explore defensive symbionts in other systems.

Giardiasis and diarrhea in dogs: Does the microbiome matter?

BACKGROUND: Giardia duodenalis (Gd) causes intestinal parasitosis. The involvement of the intestinal microbiome in determining the infection's clinical phenotype is unknown. OBJECTIVE: Investigate the fecal microbiome features in dogs with giardiasis. ANIMALS AND METHODS: Cross-sectional study, including fecal samples of kenneled dogs with Gd diagnosed by fecal Giardia antigen dot ELISA. The fecal microbial compositional characteristics and dysbiosis index (DI) were compared between diarrheic and nondiarrheic dogs. RESULTS: Fecal samples of 38 Gd-infected dogs (diarrheic, 21; nondiarrheic, 17) were included. No differences were found in Faith's phylogenic diversity and beta diversity (weighted UniFrac distances) and in specific taxa abundances at the phylum, genus, and species levels, as well as in alpha and beta diversities between diarrheic and nondiarrheic dogs, and also when divided by sex or age. Among diarrheic dogs, alpha diversity was higher in males than in females (pairwise Kruskal-Wallis, q = 0.01). Among males, fecal abundances of the genus Clostridium (W = 19) and Clostridium spiroforme species (W = 33) were higher in diarrheic compared to nondiarrheic dogs. In diarrheic dog fecal samples, Proteobacteria were more prevalent (W = 1), whereas Verrucomicrobia were less prevalent in dogs <1 year of age than in older dogs. The fecal sample DI of 19 diarrheic and 19 nondiarrheic dogs was similar (median, -0.2; range, -4.3 to 4.5 and median, -1.0; range, -4.3 to 5.8, respectively). CONCLUSIONS: The fecal microbial composition of symptomatic and asymptomatic dogs with giardiasis is similar. Based on fecal DI, giardiasis is not characterized by prominent dysbiosis. Other host and parasite characteristics might determine the severity of giardiasis in dogs.

Postnatal 14D is the Key Window for Mice Intestinal Development- An Insight from Age-Dependent Antibiotic-Mediated Gut Microbial Dysbiosis Study.

The postnatal period is one of the critical windows for the structure-function development of the gastrointestinal tract and associated mucosal immunity. Along with other constituent members, recent studies suggest the contribution of gut microbiota in maintaining host health, immunity, and development. Although the gut microbiota's role in maintaining barrier integrity is known, its function in early life development still needs to be better understood. To understand the details of gut microbiota's effects on intestinal integrity, epithelium development, and immune profile, the route of antibiotic-mediated perturbation is taken. Mice on days 7(P7D), 14(P14D), 21(P21D) and 28(P28D) are sacrificed and 16S rRNA metagenomic analysis is performed. The barrier integrity, tight junction proteins (TJPs) expression, intestinal epithelial cell (IEC) markers, and inflammatory cytokines are analyzed. Results reveal a postnatal age-related impact of gut microbiota perturbation, with a gradual increase in the relative abundance of Proteobacteria and a reduction in Bacteroidetes and Firmicutes. Significant barrier integrity disruption, reduced TJPs and IECs marker expression, and increased systemic inflammation at P14D of AVNM-treated mice are found. Moreover, the microbiota transplantation shows recolonization of Verrucomicrobia, proving a causal role in barrier functions. The investigation reveals P14D as a critical period for neonatal intestinal development, regulated by specific microbiota composition.

"Failure To Launch": Development of a Reproductive Organ Linked to Symbiotic Bacteria.

Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes. IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes. Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

Bacterioplankton seasonality in deep high-mountain lakes.

Due to global warming, shorter ice cover duration might drastically affect the ecology of lakes currently undergoing seasonal surface freezing. High-mountain lakes show snow-rich ice covers that determine contrasting conditions between ice-off and ice-on periods. We characterized the bacterioplankton seasonality in a deep high-mountain lake ice-covered for half a year. The lake shows a rich core bacterioplankton community consisting of three components: (i) an assemblage stable throughout the year, dominated by Actinobacteria, resistant to all environmental conditions; (ii) an ice-on-resilient assemblage dominating during the ice-covered period, which is more diverse than the other components and includes a high abundance of Verrucomicrobia; the deep hypolimnion constitutes a refuge for many of the typical under-ice taxa, many of which recover quickly during autumn mixing; and (iii) an ice-off-resilient assemblage, which members peak in summer in epilimnetic waters when the rest decline, characterized by a dominance of Flavobacterium, and Limnohabitans. The rich core community and low random elements compared to other relatively small cold lakes can be attributed to its simple hydrological network in a poorly-vegetated catchment, the long water-residence time (ca. 4 years), and the long ice-cover duration; features common to many headwater deep high-mountain lakes.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2-3) and moderately thermophilic conditions (50-60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including "first line of defense" mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the "second line of defense" where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

Changes in resident microbiota associated with mice susceptibility or resistance to the intestinal trematode Echinostoma caproni.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host that has been extensively used as an experimental model to study the factors that determine resistance against intestinal helminths. In E. caproni infections in mice, interleukin-25 (IL-25) plays a critical role and it is required for the resistance to infection. However, little is known on the factors that determine its production. Primary E. caproni infection in mice is characterized by the development of chronic infections and elevated worm recovery, in relation to a local Th1 response with elevated production of interferon-γ. However, partial resistance against secondary E. caproni infections in ICR (Institute of Cancer Research) mice is developed after the chemotherapeutic cure of a primary infection and the innately produced IL-25 after pharmacological treatment. In this paper, we analyse the potential role of intestinal microbiota in the production of IL-25, and the subsequent resistance to infection. For this purpose, we analysed the production of IL-25 under conditions of experimental dysbiosis and also the changes in the resident microbiota in primary infections, pharmacological curation and secondary infections. The results obtained showed that resident microbiota play a major role in the production of IL-25 and the appearance of members of the phylum Verrucomicrobia as a consequence of the curation of the primary infection could be related to the partial resistance to secondary infection.

Uncovering Lasonolide A Biosynthesis Using Genome-Resolved Metagenomics.

Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named "Candidatus Thermopylae lasonolidus," only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. IMPORTANCE While sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small quantities by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations, which causes premature chromosome condensation, blebbing, cell contraction, and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anticancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing, identified genes likely responsible for LSA synthesis in an uncultured bacterium, and assembled the symbiont's genome. These insights provide future opportunities for heterologous expression and cultivation efforts that may minimize LSA's supply problem.

Comparative Genomics and Physiology of Akkermansia muciniphila Isolates from Human Intestine Reveal Specialized Mucosal Adaptation.

Akkermansia muciniphila is a champion of mucin degradation in the human gastrointestinal tract. Here, we report the isolation of six novel strains from healthy human donors and their genomic, proteomic and physiological characterization in comparison to the type-strains A. muciniphila MucT and A. glycaniphila PytT. Complete genome sequencing revealed that, despite their large genomic similarity (>97.6%), the novel isolates clustered into two distinct subspecies of A. muciniphila: Amuc1, which includes the type-strain MucT, and AmucU, a cluster of unassigned strains that have not yet been well characterized. CRISPR analysis showed all strains to be unique and confirmed that single healthy subjects can carry more than one A. muciniphila strain. Mucin degradation pathways were strongly conserved amongst all isolates, illustrating the exemplary niche adaptation of A. muciniphila to the mucin interface. This was confirmed by analysis of the predicted glycoside hydrolase profiles and supported by comparing the proteomes of A. muciniphila strain H2, belonging to the AmucU cluster, to MucT and A. glycaniphila PytT (including 610 and 727 proteins, respectively). While some intrinsic resistance was observed among the A. muciniphila straind, none of these seem to pose strain-specific risks in terms of their antibiotic resistance patterns nor a significant risk for the horizontal transfer of antibiotic resistance determinants, opening the way to apply the type-strain MucT or these new A. muciniphila strains as next generation beneficial microbes.

Effects of Intravenous Antimicrobial Drugs on the Equine Fecal Microbiome.

Alterations in the gastrointestinal microbiota after antimicrobial therapy in horses can result in loss of colonization resistance and changes in bacterial metabolic function. It is hypothesized that these changes facilitate gastrointestinal inflammation, pathogen expansion and the development of diarrhea. The objectives of this study were to determine the effect of intravenous administration of antimicrobial drugs (ceftiofur, enrofloxacin, oxytetracycline) on equine fecal bacterial communities over time, to investigate whether those changes are detectable after 5 days of treatment and whether they persist over time (30 days). Sixteen horses were randomly assigned into 4 treatment groups: group 1 (enrofloxacin, n = 4); group 2 (ceftiofur sodium, n = 4); group 3 (oxytetracycline, n = 4); group 4 (0.9% saline solution, placebo, n = 4). Antimicrobial therapy was administered for 5 days. Fecal samples were obtained before (day 0) and at 3, 5 and 30 days of the study period. Bacterial DNA was amplified using specific primers to the hypervariable region V1−V3 of the 16S rRNA gene using a 454 FLX-Titanium pyrosequencer. Antimicrobial therapy failed to cause any changes in physical examination parameters, behavior, appetite or fecal output or consistency throughout the study in any horse. There was a significant effect of treatment on alpha diversity indices (richness) over the treatment interval for ceftiofur on days 0 vs. 3 (p < 0.05), but not for other antimicrobials (p > 0.05). Microbial composition was significantly different (p < 0.05) across treatment group and day, but not for interactions between treatment and day, regardless of taxonomic level and beta-diversity distance metric. The most significant antimicrobial effects on relative abundance were noted after intravenous administration of ceftiofur and enrofloxacin. The relative abundance of Fibrobacteres was markedly lower on day 3 compared to other days in the ceftiofur and enrofloxacin treatment groups. There was an increase in Clostridia and Lachnospiraceae from day 0 to days 3 and 5 in ceftiofur and enrofloxacin treated groups. These findings showed the negative effect of antimicrobial drugs on bacterial communities associated with gut health (Fibrobacteres and Lachnospiraceae) and indicate that changes in specific taxa could predispose horses to gastrointestinal inflammation and the development of diarrhea.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as "Candidatus Synoicihabitans palmerolidicus." The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium. IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, "Candidatus Synoicihabitans palmerolidicus," in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer's genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Peat-Inhabiting Verrucomicrobia of the Order Methylacidiphilales Do Not Possess Methanotrophic Capabilities.

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5-5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83-86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.

Altered milk yield and rumen microbial abundance in response to concentrate supplementation during the cold season in Tibetan sheep

BACKGROUND Weight loss and decline of milk yield in Tibetan sheep was a challenge for the dairy industry in Qinghai-Tibet Plateau, which were considered to be caused by underfeeding of the sheep during the harsh winter. The objective of this study was to assess the role of feed supplementation in the milk performance and rumen microbiome of ewes under forage-based diets. Based on parity, milking period, milk yield, and body weight, ten 1.5-yr-old ewes were allocated randomly into two groups. One group of ewes was fed no supplement Control group (CON) and the other group was fed with concentrate feed supplement (Treatment group, T). Individual milk yield was determined daily; both the milk composition and rumen bacterial characteristics were analyzed after the end of feeding trials. RESULTS Results showed that lactose in the milk of the CON group was significantly lower (P < 0.05) than that of the T group at days 30 and 60. Milk yield in the T group was greater than in the CON group at day 30 (P < 0.05). Additionally, the dominant ruminal bacteria (phyla Bacteroidetes, Firmicutes, and Verrucomicrobia) were shared by both groups through 16S rRNA gene pyrosequencing. Greater relative abundance of Bacteroidales RF16 group in family level, Victivallales in order level, Lentisphaeria in class level, and Lachnospiraceae bacterium in species level were observed in the T group than in the CON group (P < 0.05). CONCLUSIONS These results demonstrated that supplementation of concentrate in the cold season improved milk lactose yield and milk production, and the rumen microbial abundance of Tibetan sheep.

Crystal structure of a homotrimeric verrucomicrobial exo-ß-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-ß-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-ß-1,4-mannosidase [EC 3.2.1.25] that is highly specific for ß-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Šresolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family ß-galactosidases and the GH164-family exo-ß-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 ß-galactosidase-type homotrimeric structure.

High-fat diet prevents the development of autoimmune diabetes in NOD mice.

AIMS: Type 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate the beta-cell autoimmune destruction. The rate of childhood obesity has risen in parallel to the proportion of T1D, suggesting high-fat diet (HFD)/obesity as potential environmental triggers for autoimmune diabetes. To explore this, non-obese diabetic (NOD) mice were subjected to HFD and monitored for the development of diabetes, insulitis and beta-cell stress. MATERIALS AND METHODS: Four-week-old female NOD mice were placed on HFD (HFD-NOD) or standard chow-diet. Blood glucose was monitored weekly up to 40 weeks of age, and glucose- and insulin-tolerance tests performed at 4, 10 and 15 weeks. Pancreata and islets were analysed for insulin secretion, beta-cell mass, inflammation, insulitis and endoplasmic reticulum stress markers. Immune cell levels were measured in islets and spleens. Stool microbiome was analysed at age 4, 8 and 25 weeks. RESULTS: At early ages, HFD-NOD mice showed a significant increase in body weight, glucose intolerance and insulin resistance; but paradoxically, they were protected from developing diabetes. This was accompanied by increased insulin secretion and beta-cell mass, decreased insulitis, increased splenic T-regulatory cells and altered stool microbiome. CONCLUSIONS: This study shows that HFD protects NOD mice from autoimmune diabetes and preserves beta-cell mass and function through alterations in gut microbiome, increased T-regulatory cells and decreased insulitis. Further studies into the exact mechanism of HFD-mediated prevention of diabetes in NOD mice could potentially lead to interventions to prevent or delay T1D development in humans.

Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island.

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of "Candidatus Methylacidithermus pantelleriae" PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, "Ca. Methylacidithermus pantelleriae" might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.

Genotypic and Phenotypic Diversity among Human Isolates of Akkermansia muciniphila.

The mucophilic anaerobic bacterium Akkermansia muciniphila is a prominent member of the gastrointestinal (GI) microbiota and the only known species of the Verrucomicrobia phylum in the mammalian gut. A high prevalence of A. muciniphila in adult humans is associated with leanness and a lower risk for the development of obesity and diabetes. Four distinct A. muciniphila phylogenetic groups have been described, but little is known about their relative abundance in humans or how they impact human metabolic health. In this study, we isolated and characterized 71 new A. muciniphila strains from a cohort of children and adolescents undergoing treatment for obesity. Based on genomic and phenotypic analysis of these strains, we found several phylogroup-specific phenotypes that may impact the colonization of the GI tract or modulate host functions, such as oxygen tolerance, adherence to epithelial cells, iron and sulfur metabolism, and bacterial aggregation. In antibiotic-treated mice, phylogroups AmIV and AmII outcompeted AmI strains. In children and adolescents, AmI strains were most prominent, but we observed high variance in A. muciniphila abundance and single phylogroup dominance, with phylogroup switching occurring in a small subset of patients. Overall, these results highlight that the ecological principles determining which A. muciniphila phylogroup predominates in humans are complex and that A. muciniphila strain genetic and phenotypic diversity may represent an important variable that should be taken into account when making inferences as to this microbe's impact on its host's health.IMPORTANCE The abundance of Akkermansia muciniphila in the gastrointestinal (GI) tract is linked to multiple positive health outcomes. There are four known A. muciniphila phylogroups, yet the prevalence of these phylogroups and how they vary in their ability to influence human health is largely unknown. In this study, we performed a genomic and phenotypic analysis of 71 A. muciniphila strains and identified phylogroup-specific traits such as oxygen tolerance, adherence, and sulfur acquisition that likely influence colonization of the GI tract and differentially impact metabolic and immunological health. In humans, we observed that single Akkermansia phylogroups predominate at a given time but that the phylotype can switch in an individual. This collection of strains provides the foundation for the functional characterization of A. muciniphila phylogroup-specific effects on the multitude of host outcomes associated with Akkermansia colonization, including protection from obesity, diabetes, colitis, and neurological diseases, as well as enhanced responses to cancer immunotherapies.