Phytoplankton exudates and lysates support distinct microbial consortia with specialized metabolic and ecophysiological traits.

Blooms of marine phytoplankton fix complex pools of dissolved organic matter (DOM) that are thought to be partitioned among hundreds of heterotrophic microbes at the base of the food web. While the relationship between microbial consumers and phytoplankton DOM is a key component of marine carbon cycling, microbial loop metabolism is largely understood from model organisms and substrates. Here, we took an untargeted approach to measure and analyze partitioning of four distinct phytoplankton-derived DOM pools among heterotrophic populations in a natural microbial community using a combination of ecogenomics, stable isotope probing (SIP), and proteomics. Each 13C-labeled exudate or lysate from a diatom or a picocyanobacterium was preferentially assimilated by different heterotrophic taxa with specialized metabolic and physiological adaptations. Bacteroidetes populations, with their unique high-molecular-weight transporters, were superior competitors for DOM derived from diatom cell lysis, rapidly increasing growth rates and ribosomal protein expression to produce new relatively high C:N biomass. Proteobacteria responses varied, with relatively low levels of assimilation by Gammaproteobacteria populations, while copiotrophic Alphaproteobacteria such as the Roseobacter clade, with their diverse array of ABC- and TRAP-type transporters to scavenge monomers and nitrogen-rich metabolites, accounted for nearly all cyanobacteria exudate assimilation and produced new relatively low C:N biomass. Carbon assimilation rates calculated from SIP data show that exudate and lysate from two common marine phytoplankton are being used by taxonomically distinct sets of heterotrophic populations with unique metabolic adaptations, providing a deeper mechanistic understanding of consumer succession and carbon use during marine bloom events.

Recovery and Community Succession of the Zostera marina Rhizobiome after Transplantation.

Seagrasses can form mutualisms with their microbiomes that facilitate the exchange of energy sources, nutrients, and hormones and ultimately impact plant stress resistance. Little is known about community succession within the belowground seagrass microbiome after disturbance and its potential role in the plant's recovery after transplantation. We transplanted Zostera marina shoots with and without an intact rhizosphere and cultivated plants for 4 weeks while characterizing microbiome recovery and effects on plant traits. Rhizosphere and root microbiomes were compositionally distinct, likely representing discrete microbial niches. Furthermore, microbiomes of washed transplants were initially different from those of sod transplants and recovered to resemble an undisturbed state within 14 days. Conspicuously, changes in the microbial communities of washed transplants corresponded with changes in the rhizosphere sediment mass and root biomass, highlighting the strength and responsive nature of the relationship between plants, their microbiome, and the environment. Potential mutualistic microbes that were enriched over time include those that function in the cycling and turnover of sulfur, nitrogen, and plant-derived carbon in the rhizosphere environment. These findings highlight the importance and resilience of the seagrass microbiome after disturbance. Consideration of the microbiome will have meaningful implications for habitat restoration practices.IMPORTANCE Seagrasses are important coastal species that are declining globally, and transplantation can be used to combat these declines. However, the bacterial communities associated with seagrass rhizospheres and roots (the microbiome) are often disturbed or removed completely prior to transplantation. The seagrass microbiome benefits seagrasses through metabolite, nutrient, and phytohormone exchange and contributes to the ecosystem services of seagrass meadows by cycling sulfur, nitrogen, and carbon. This experiment aimed to characterize the importance and resilience of the seagrass belowground microbiome by transplanting Zostera marina with and without intact rhizospheres and tracking microbiome and plant morphological recovery over 4 weeks. We found the seagrass microbiome to be resilient to transplantation disturbance, recovering after 14 days. Additionally, microbiome recovery was linked with seagrass morphology, coinciding with increases in the rhizosphere sediment mass and root biomass. The results of this study can be used to include microbiome responses in informing future restoration work.

Comparative genomic analysis of Vibrios yields insights into genes associated with virulence towards C. gigas larvae.

BACKGROUND: Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae. RESULTS: V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae. CONCLUSIONS: These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

Sipros Ensemble improves database searching and filtering for complex metaproteomics.

Motivation: Complex microbial communities can be characterized by metagenomics and metaproteomics. However, metagenome assemblies often generate enormous, and yet incomplete, protein databases, which undermines the identification of peptides and proteins in metaproteomics. This challenge calls for increased discrimination of true identifications from false identifications by database searching and filtering algorithms in metaproteomics. Results: Sipros Ensemble was developed here for metaproteomics using an ensemble approach. Three diverse scoring functions from MyriMatch, Comet and the original Sipros were incorporated within a single database searching engine. Supervised classification with logistic regression was used to filter database searching results. Benchmarking with soil and marine microbial communities demonstrated a higher number of peptide and protein identifications by Sipros Ensemble than MyriMatch/Percolator, Comet/Percolator, MS-GF+/Percolator, Comet & MyriMatch/iProphet and Comet & MyriMatch & MS-GF+/iProphet. Sipros Ensemble was computationally efficient and scalable on supercomputers. Availability and implementation: Freely available under the GNU GPL license at http://sipros.omicsbio.org. Contact: cpan@utk.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

BACKGROUND: Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. RESULTS: The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. CONCLUSION: Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

Chloroflexi CL500-11 Populations That Predominate Deep-Lake Hypolimnion Bacterioplankton Rely on Nitrogen-Rich Dissolved Organic Matter Metabolism and C1 Compound Oxidation.

The Chloroflexi CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides. In situ expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat.

Corrigendum: Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

[This corrects the article DOI: 10.3389/fimmu.2024.1380089.].

Proteomic stable isotope probing with an upgraded Sipros algorithm for improved identification and quantification of isotopically labeled proteins.

BACKGROUND: Proteomic stable isotope probing (SIP) is used in microbial ecology to trace a non-radioactive isotope from a labeled substrate into de novo synthesized proteins in specific populations that are actively assimilating and metabolizing the substrate in a complex microbial community. The Sipros algorithm is used in proteomic SIP to identify variably labeled proteins and quantify their isotopic enrichment levels (atom%) by performing enrichment-resolved database searching. RESULTS: In this study, Sipros was upgraded to improve the labeled protein identification, isotopic enrichment quantification, and database searching speed. The new Sipros 4 was compared with the existing Sipros 3, Calisp, and MetaProSIP in terms of the number of identifications and the accuracy and precision of atom% quantification on both the peptide and protein levels using standard E. coli cultures with 1.07 atom%, 2 atom%, 5 atom%, 25 atom%, 50 atom%, and 99 atom% 13C enrichment. Sipros 4 outperformed Calisp and MetaProSIP across all samples, especially in samples with ≥ 5 atom% 13C labeling. The computational speed on Sipros 4 was > 20 times higher than Sipros 3 and was on par with the overall speed of Calisp- and MetaProSIP-based pipelines. Sipros 4 also demonstrated higher sensitivity for the detection of labeled proteins in two 13C-SIP experiments on a real-world soil community. The labeled proteins were used to trace 13C from 13C-methanol and 13C-labeled plant exudates to the consuming soil microorganisms and their newly synthesized proteins. CONCLUSION: Overall, Sipros 4 improved the quality of the proteomic SIP results and reduced the computational cost of SIP database searching, which will make proteomic SIP more useful and accessible to the border community. Video Abstract.

Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

Introduction: The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination. Methods: Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment. Results: Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%. Discussion: These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Biosponge-Encased Placental Stem Cells for Volumetric Muscle Loss Repair.

OBJECTIVE: Volumetric muscle loss (VML) leads to permanent muscle mass and functional impairments. While mesenchymal stromal cells (MSCs) and their secreted factors can aid muscle regeneration, MSCs exhibit limited persistence in injured tissue post-transplantation. Human placenta-derived stem cells (hPDSCs), sharing surface markers with MSCs, demonstrate superior regenerative potential due to their fetal origin. Previously, a biosponge (BS) scaffold was shown to augment muscle regeneration post-VML. This study aims to co-apply BS therapy and hPDSCs to further enhance muscle recovery following VML. APPROACH: A VML defect was created by removing ~20% of the tibialis anterior muscle mass in male Lewis rats. Injured muscles were either left untreated or treated with BS or BS-encapsulated hPDSCs cultured under normoxic or hypoxic conditions. On day 28 post-injury, peak isometric torque was measured, and the muscle was harvested for analysis. RESULTS: BS encapsulated hPDSCs subjected to hypoxic preconditioning persisted in larger quantities and enhanced muscle mass at day 28 post-injury. BS encapsulated hPDSCs cultured under normoxic or hypoxic conditions increased small myofibers (<500 µm2) percentage, MyoD protein expression, and both pro- and anti-inflammatory macrophage marker expression. BS encapsulated hPDSCs also reduced fibrosis and BS remodeling rate. INNOVATION: This study is the first to examine the therapeutic effects of hPDSCs in a rat VML model. A BS carrier and hypoxic preconditioning were investigated to mitigate low cell survival post-implantation. CONCLUSION: hPDSCs augment the regenerative effect of BS. Combining hPDSCs and BS emerges as a promising strategy worthy of further investigation.

Genomic and epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse survival in lethal prostate cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSIs) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood. EXPERIMENTAL DESIGN: We applied targeted cell-free DNA sequencing to 126 mCRPC patients from three academic cancer centers, and separately performed genome-wide cell-free DNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome-positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 mCRPC patients, respectively, to develop and validate a stem-like signature, which we inferred from cell-free DNA. RESULTS: Targeted cell-free DNA sequencing detected AR/enhancer alterations prior to first-line ARSIs which correlated with significantly worse PFS (p = 0.01; HR = 2.12) and OS (p = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (p < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cell-free DNA revealed enrichment for stemness-associated transcription factors in lethal mCRPC patients. The resulting stemness signature was then validated in a completely held-out cohort of 80 mCRPC patients profiled by tumor RNA sequencing. CONCLUSIONS: We analyzed a total of 220 mCRPC patients, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.

Femtosecond electron-lattice thermalization dynamics in a gold film probed by pulsed surface plasmon resonance.

The dynamics of electronic excitations and their relaxation in a gold film is studied on the femtosecond time scale with a pump-probe technique. For the pump beam we use pulses with wavelengths centered at 800 nm, 400 nm or both. The surface plasmon resonance (SPR) in Kretschmann's configuration is used as a sensitive and fast-response probe of the dynamics of the dielectric properties of the gold film. The quantity that is monitored is the intensity of the reflected light at an incidence angle close to the SPR. With changes of the dielectric properties induced by the pump beam and during subsequent relaxation, the amount of the reflected light of the probe beam, sent with a variable delay, also changes, thus providing information on the temporal characteristics of the thermalization process. Special features of SPR probing with short pulses are also accounted for in this work. The thermalization of the electronic subsystem and energy transfer to the lattice are discussed in connection with the two-temperature relaxation model that takes into account temperature dependences of the electronic heat capacity and the electron-phonon coupling.

Proteogenomic basis for ecological divergence of closely related bacteria in natural acidophilic microbial communities.

Bacterial species concepts are controversial. More widely accepted is the need to understand how differences in gene content and sequence lead to ecological divergence. To address this relationship in ecosystem context, we investigated links between genotype and ecology of two genotypic groups of Leptospirillum group II bacteria in comprehensively characterized, natural acidophilic biofilm communities. These groups share 99.7% 16S rRNA gene sequence identity and 95% average amino acid identity between their orthologs. One genotypic group predominates during early colonization, and the other group typically proliferates in later successional stages, forming distinct patches tens to hundreds of micrometers in diameter. Among early colonizing populations, we observed dominance of five genotypes that differed from each other by the extent of recombination with the late colonizing type. Our analyses suggest that the specific recombinant variant within the early colonizing group is selected for by environmental parameters such as temperature, consistent with recombination as a mechanism for ecological fine tuning. Evolutionary signatures, and strain-resolved expression patterns measured via mass spectrometry-based proteomics, indicate increased cobalamin biosynthesis, (de)methylation, and glycine cleavage in the late colonizer. This may suggest environmental changes within the biofilm during development, accompanied by redirection of compatible solutes from osmoprotectants toward metabolism. Across 27 communities, comparative proteogenomic analyses show that differential regulation of shared genes and expression of a small subset of the approximately 15% of genes unique to each genotype are involved in niche partitioning. In summary, the results show how subtle genetic variations can lead to distinct ecological strategies.

Woronichinia naegeliana: A common nontoxigenic component of temperate freshwater cyanobacterial blooms with 30% of its genome in transposons.

Monitoring in the U.S. state of Washington across the period 2007-2019 showed that Woronichinia has been present in many lakes state-wide. This cyanobacterium was commonly dominant or sub-dominant in cyanobacterial blooms in the wet temperate region west of the Cascade Mountains. In these lakes, Woronichinia often co-existed with Microcystis, Dolichospermum and Aphanizomenon flos-aquae and the cyanotoxin microcystin has often been present in those blooms, although it has not been known whether Woronichinia is a toxin producer. We report the first complete genome of Woronichinia naegeliana WA131, assembled from the metagenome of a sample collected from Wiser Lake, Washington, in 2018. The genome contains no genes for cyanotoxin biosynthesis or taste-and-odor compounds, but there are biosynthetic gene clusters for other bioactive peptides, including anabaenopeptins, cyanopeptolins, microginins and ribosomally produced, post-translationally modified peptides. Genes for photosynthesis, nutrient acquisition, vitamin synthesis and buoyancy that are typical of bloom-forming cyanobacteria are present, although nitrate and nitrite reductase genes are conspicuously absent. However, the 7.9 Mbp genome is 3-4 Mbp larger than those of the above-mentioned frequently co-existing cyanobacteria. The increased genome size is largely due to an extraordinary number of insertion sequence elements (transposons), which account for 30.3% of the genome and many of which are present in multiple copies. The genome contains a relatively large number of pseudogenes, 97% of which are transposase genes. W. naegeliana WA131 thus seems to be able to limit the potentially deleterious effects of high rates of recombination and transposition to the mobilome fraction of its genome.

Genomic and epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse survival in AR -altered lethal prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor (AR)-targeted agents is often lethal. Unfortunately, biomarkers for this deadly disease remain under investigation, and underpinning mechanisms are ill-understood. Here, we applied deep sequencing to ∼100 mCRPC patients prior to the initiation of first-line AR-targeted therapy, which detected AR /enhancer alterations in over a third of patients, which correlated with lethality. To delve into the mechanism underlying why these patients with cell-free AR /enhancer alterations developed more lethal prostate cancer, we next performed genome-wide cell-free DNA epigenomics. Strikingly, we found that binding sites for transcription factors associated with developmental stemness were nucleosomally more accessible. These results were corroborated using cell-free DNA methylation data, as well as tumor RNA sequencing from a held-out cohort of mCRPC patients. Thus, we validated the importance of AR /enhancer alterations as a prognostic biomarker in lethal mCRPC, and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.

Metal affinity enrichment increases the range and depth of proteome identification for extracellular microbial proteins.

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as "hypothetical" were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

Heterotrophic archaea contribute to carbon cycling in low-pH, suboxic biofilm communities.

Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria- to Archaea-dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata, and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.

Genome sequence of freshwater nontoxigenic Limnoraphis associated with microcystin-producing blooms.

A sample from a 2019 cyanobacterial bloom in a freshwater reservoir in eastern Oregon, USA, was used to produce a metagenome from which the complete, circular 7.3 Mbp genome of Limnoraphis sp. WC205 was assembled. The Limnoraphis sp. WC205 genome contains gas vesicle genes, genes for N2-fixation and genes for both phycocyanin- and phycoerythrin-containing phycobilisomes. Limnoraphis was present in Willow Creek Reservoir throughout the summer and fall, coexisting with various other cyanobacteria in blooms that were associated with microcystin. The absence of cyanotoxin genes from the Limnoraphis sp. WC205 genome showed this cyanobacterium to be non-toxigenic, although it is predicted to produce cyanobactins closely related to Microcystis aeruginosa microcyclamides. DNA sequence corresponding to the Microcystis mcyG gene identified Microcystis as the microcystin producer in this lake.

7-epi-cylindrospermopsin and microcystin producers among diverse Anabaena/Dolichospermum/Aphanizomenon CyanoHABs in Oregon, USA.

Several genomes of Nostocales ADA clade members from the US Pacific Northwest were recently sequenced. Biosynthetic genes for microcystin, cylindrospermopsin or anatoxin-a were present in 7 of the 15 Dolichospermum/Anabaena strains and none of the 5 Aphanizomenon flos-aquae (AFA) strains. Toxin analyses (ELISA and LC-MS/MS) were conducted to quantitate and identify microcystin (MC) and cylindrospermopsin (CYN) congeners/analogs in samples dominated by Dolichospermum spp. of known genome sequence. MC-LR was the main congener produced by Dolichospermum spp. from Junipers Reservoir, Lake Billy Chinook and Odell Lake, while a congener provisionally identified as [Dha7]MC-HtyR was produced by a Dolichospermum sp. in Detroit Reservoir. A second Dolichospermum sp. from Detroit Reservoir was found to produce 7-epi-CYN, with 7-deoxy-CYN also present, but no CYN. The monitoring history of each of these lakes indicates the capacity for high levels of cyanotoxins during periods when Dolichospermum spp. are the dominant cyanobacteria. The diversity of ADA strains found in the US Pacific NW emphasizes the importance of these cyanobacteria as potentially toxic HAB formers in this temperate climatic region. Our results linking congener and genetic identity add data points that will help guide development of improved tools for predicting congener specificity from cyanotoxin gene sequences.

Islet autoantibody seroconversion in type-1 diabetes is associated with metagenome-assembled genomes in infant gut microbiomes.

The immune system of some genetically susceptible children can be triggered by certain environmental factors to produce islet autoantibodies (IA) against pancreatic ß cells, which greatly increases their risk for Type-1 diabetes. An environmental factor under active investigation is the gut microbiome due to its important role in immune system education. Here, we study gut metagenomes that are de-novo-assembled in 887 at-risk children in the Environmental Determinants of Diabetes in the Young (TEDDY) project. Our results reveal a small set of core protein families, present in >50% of the subjects, which account for 64% of the sequencing reads. Time-series binning generates 21,536 high-quality metagenome-assembled genomes (MAGs) from 883 species, including 176 species that hitherto have no MAG representation in previous comprehensive human microbiome surveys. IA seroconversion is positively associated with 2373 MAGs and negatively with 1549 MAGs. Comparative genomics analysis identifies lipopolysaccharides biosynthesis in Bacteroides MAGs and sulfate reduction in Anaerostipes MAGs as functional signatures of MAGs with positive IA-association. The functional signatures in the MAGs with negative IA-association include carbohydrate degradation in lactic acid bacteria MAGs and nitrate reduction in Escherichia MAGs. Overall, our results show a distinct set of gut microorganisms associated with IA seroconversion and uncovered the functional genomics signatures of these IA-associated microorganisms.