Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility.

Aquatic bacteria frequently are divided into lifestyle categories oligotroph or copiotroph. Oligotrophs have proportionately fewer transcriptional regulatory genes than copiotrophs and are generally non-motile/chemotactic. We hypothesized that the absence of chemotaxis/motility in oligotrophs prevents them from occupying nutrient patches long enough to benefit from transcriptional regulation. We first confirmed that marine oligotrophs are generally reduced in genes for transcriptional regulation and motility/chemotaxis. Next, using a non-motile oligotroph (Ca. Pelagibacter st. HTCC7211), a motile copiotroph (Alteromonas macleodii st. HOT1A3), and [14 C]l-alanine, we confirmed that l-alanine catabolism is not transcriptionally regulated in HTCC7211 but is in HOT1A3. We then found that HOT1A3 took 2.5-4 min to initiate l-alanine oxidation at patch l-alanine concentrations, compared to <30 s for HTCC7211. By modelling cell trajectories, we predicted that, in most scenarios, non-motile cells spend <2 min in patches, compared to >4 min for chemotactic/motile cells. Thus, the time necessary for transcriptional regulation to initiate prevents transcriptional regulation from being beneficial for non-motile oligotrophs. This is supported by a mechanistic model we developed, which predicted that HTCC7211 cells with transcriptional regulation of l-alanine metabolism would produce 12% of their standing ATP stock upon encountering an l-alanine patch, compared to 880% in HTCC7211 cells without transcriptional regulation.

Metabolism of key atmospheric volatile organic compounds by the marine heterotrophic bacterium Pelagibacter HTCC1062 (SAR11).

Plants and phytoplankton are natural sources of the volatile organic compounds (VOCs) acetone and isoprene, which are reactive and can alter atmospheric chemistry. In earlier research we reported that, when co-cultured with a diatom, the marine bacterium Pelagibacter (strain HTCC1062; 'SAR11 clade') reduced the concentration of compounds tentatively identified as acetone and isoprene. In this study, experiments with Pelagibacter monocultures confirmed that these cells are capable of metabolizing acetone and isoprene at rates similar to bacterial communities in seawater and high enough to consume substantial fractions of the total marine acetone and isoprene budgets if extrapolated to global SAR11 populations. Homologues of an acetone/cyclohexanone monooxygenase were identified in the HTCC1062 genome and in the genomes of a wide variety of other abundant marine taxa, and were expressed at substantial levels (c. 10-4 of transcripts) across TARA oceans metatranscriptomes from ocean surface samples. The HTCC1062 genome lacks the canonical isoprene degradation pathway, suggesting an unknown alternative biochemical pathway is used by these cells for isoprene uptake. Fosmidomycin, an inhibitor of bacterial isoprenoid biosynthesis, blocked HTCC1062 growth, but the cells were rescued when isoprene was added to the culture, indicating SAR11 cells may be capable of synthesizing isoprenoid compounds from exogenous isoprene.

Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation.

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and -utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals.

Multi-Stimuli-Responsive Janus Hollow Polydopamine Nanotubes.

A tubular-shaped Janus nanoparticle based on polydopamine that responds to near-infrared, magnetic, and pH stimuli is reported. The robust tubular polydopamine structure was obtained by optimizing the halloysite template-to-dopamine ratio during synthesis. The inner and outer surfaces of the tube were exposed at different steps of the template-sonication--etching process, enabling the differential surface modification of these surfaces. Poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAM) were grafted to the outer and inner surface of the nanotube, respectively. The PEG-coated surface limited aggregation of the nanoparticles at elevated temperatures. The PNIPAM-coated interior enhanced doxorubicin loading and endowed the nanoparticle with temperature-responsive behavior. The deposition of precipitated Fe3O4 nanoparticles further modified the nanoparticles. The resulting magnetic Janus nanoparticles responded to pH, temperature, and magnetic fields. Temperature changes could be induced by near-infrared laser, and all three stimuli were found to influence release rates of adsorbed doxorubicin from the nanoparticles. The interaction of the stimuli on release kinetics was elucidated using a linear mixed model; reduced pH and NIR irradiation enhanced release while applying a static magnetic field retarded release. Furthermore, the mechanism was shifted toward Fickian behavior by applying a static magnetic field and low pH conditions. However, NIR irradiation only shifted the behavior toward Fickian behavior at low pH.

Pan-tissue transcriptome analysis of long noncoding RNAs in the American beaver Castor canadensis.

BACKGROUND: Long noncoding RNAs (lncRNAs) have roles in gene regulation, epigenetics, and molecular scaffolding and it is hypothesized that they underlie some mammalian evolutionary adaptations. However, for many mammalian species, the absence of a genome assembly precludes the comprehensive identification of lncRNAs. The genome of the American beaver (Castor canadensis) has recently been sequenced, setting the stage for the systematic identification of beaver lncRNAs and the characterization of their expression in various tissues. The objective of this study was to discover and profile polyadenylated lncRNAs in the beaver using high-throughput short-read sequencing of RNA from sixteen beaver tissues and to annotate the resulting lncRNAs based on their potential for orthology with known lncRNAs in other species. RESULTS: Using de novo transcriptome assembly, we found 9528 potential lncRNA contigs and 187 high-confidence lncRNA contigs. Of the high-confidence lncRNA contigs, 147 have no known orthologs (and thus are putative novel lncRNAs) and 40 have mammalian orthologs. The novel lncRNAs mapped to the Oregon State University (OSU) reference beaver genome with greater than 90% sequence identity. While the novel lncRNAs were on average shorter than their annotated counterparts, they were similar to the annotated lncRNAs in terms of the relationships between contig length and minimum free energy (MFE) and between coverage and contig length. We identified beaver orthologs of known lncRNAs such as XIST, MEG3, TINCR, and NIPBL-DT. We profiled the expression of the 187 high-confidence lncRNAs across 16 beaver tissues (whole blood, brain, lung, liver, heart, stomach, intestine, skeletal muscle, kidney, spleen, ovary, placenta, castor gland, tail, toe-webbing, and tongue) and identified both tissue-specific and ubiquitous lncRNAs. CONCLUSIONS: To our knowledge this is the first report of systematic identification of lncRNAs and their expression atlas in beaver. LncRNAs-both novel and those with known orthologs-are expressed in each of the beaver tissues that we analyzed. For some beaver lncRNAs with known orthologs, the tissue-specific expression patterns were phylogenetically conserved. The lncRNA sequence data files and raw sequence files are available via the web supplement and the NCBI Sequence Read Archive, respectively.

Sulforaphane Bioavailability and Chemopreventive Activity in Men Presenting for Biopsy of the Prostate Gland: A Randomized Controlled Trial.

Previous studies suggest compounds such as sulforaphane (SFN) derived from cruciferous vegetables may prevent prostate cancer development and progression. This study evaluated the effect of broccoli sprout extract (BSE) supplementation on blood histone deacetylase (HDAC) activity, prostate RNA gene expression, and tissue biomarkers (histone H3 lysine 18 acetylation (H3K18ac), HDAC3, HDAC6, Ki67, and p21). A total of 98 men scheduled for prostate biopsy were allocated into either BSE (200 µmol daily) or a placebo in our double-blind, randomized controlled trial. We used nonparametric tests to evaluate the differences of blood HDAC activity and prostate tissue immunohistochemistry biomarkers between treatment groups. Further, we performed RNA-Seq analysis on the prostate biopsies and identified 40 differentially expressed genes correlated with BSE treatment, including downregulation of two genes previously implicated in prostate cancer development, AMACR and ARLNC1. Although urine and plasma SFN isothiocyanates and individual SFN metabolites were statistically higher in the treatment group, our results did not show a significant difference in HDAC activity or prostate tissue biomarkers. This study indicates BSE supplementation correlates with changes in gene expression but not with several other prostate cancer biomarkers. More research is required to fully understand the chemopreventive effects of BSE supplementation on prostate cancer.

Tropical soils are a reservoir for fluorescent Pseudomonas spp. biodiversity.

Fluorescent Pseudomonas spp. are widely studied for their beneficial activities to plants. To explore the genetic diversity of Pseudomonas spp. in tropical regions, we collected 76 isolates from a Brazilian soil. Genomes were sequenced and compared to known strains, mostly collected from temperate regions. Phylogenetic analyses classified the isolates in the P. fluorescens (57) and P. putida (19) groups. Among the isolates in the P. fluorescens group, most (37) were classified in the P. koreensis subgroup and two in the P. jessenii subgroup. The remaining 18 isolates fell into two phylogenetic subclades distinct from currently recognized P. fluorescens subgroups, and probably represent new subgroups. Consistent with their phylogenetic distance from described subgroups, the genome sequences of strains in these subclades are asyntenous to the genome sequences of members of their neighbour subgroups. The tropical isolates have several functional genes also present in known fluorescent Pseudomonas spp. strains. However, members of the new subclades share exclusive genes not detected in other subgroups, pointing to the potential for novel functions. Additionally, we identified 12 potential new species among the 76 isolates from the tropical soil. The unexplored diversity found in the tropical soil is possibly related to biogeographical patterns.

Genome variations between rhizosphere and bulk soil ecotypes of a Pseudomonas koreensis population.

Bulk soil and rhizosphere are soil compartments selecting different microbial communities. However, it is unknown whether this selection also can change the genome content of specific bacterial taxa, splitting a population in distinct ecotypes. To answer this question we compared the genome sequences of 53 isolates obtained from sugarcane rhizosphere (28) and bulk soil (25). These isolates were previously classified in the Pseudomonas koreensis subgroup of the P. fluorescens complex. Phylogenomics showed a trend of separation between bulk soil and rhizosphere isolates. Discriminant analysis of principal components (DAPC) identified differences in the accessory genome of rhizosphere and bulk soil sub-populations. We found significant changes in gene frequencies distinguishing rhizosphere from bulk soil ecotypes, for example, enrichment of phosphatases and xylose utilization (xut) genes, respectively. Phenotypic assays and deletion of xutA gene indicated that accumulation of xut genes in the bulk soil sub-population provided a higher growth capacity in a d-xylose medium, supporting the corresponding genomic differences. Despite the clear differences distinguishing the two ecotypes, all 53 isolates were classified in a single 16S rRNA gene OTU. Collectively, our results revealed that the gene pool and ecological behavior of a bacterial population can be different for ecotypes living in neighbouring soil habitats.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites.

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch-like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Whole cell affinity for 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) in the marine bacterium Candidatus Pelagibacter st. HTCC7211 explains marine dissolved HMP concentrations.

Vitamin B1 is a universally required coenzyme in carbon metabolism. However, most marine microorganisms lack the complete biosynthetic pathway for this compound and must acquire thiamin, or precursor molecules, from the dissolved pool. The most common version of Vitamin B1 auxotrophy is for thiamin's pyrimidine precursor moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Frequent HMP auxotrophy in plankton and vanishingly low dissolved concentrations (approximately 0.1-50 pM) suggest that high-affinity HMP uptake systems are responsible for maintaining low ambient HMP concentrations. We used tritium-labelled HMP to investigate HMP uptake mechanisms and kinetics in cell cultures of Candidatus Pelagibacter st. HTCC7211, a representative of the globally distributed and highly abundant SAR11 clade. A single protein, the sodium solute symporter ThiV, which is conserved across SAR11 genomes, is the likely candidate for HMP transport. Experimental evidence indicated transport specificity for HMP and mechanistically complex, high-affinity HMP uptake kinetics. Km values ranged from 9.5 pM to 1.2 nM and were dramatically lower when cells were supplied with a carbon source. These results suggest that HMP uptake in HTCC7211 is subject to complex regulation and point to a strategy for high-affinity uptake of this essential growth factor that can explain natural HMP levels in seawater.

Ferric-pyoverdine recognition by Fpv outer membrane proteins of Pseudomonas protegens Pf-5.

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp.

pA506, a conjugative plasmid of the plant epiphyte Pseudomonas fluorescens A506.

Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOBP6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae. The origin of vegetative replication (oriV) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae, but pA506 lacks repA, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.

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.

Identification of rare microbial colonizers of plastic materials incubated in a coral reef environment.

Plastic waste accumulation in marine environments has complex, unintended impacts on ecology that cross levels of community organization. To measure succession in polyolefin-colonizing marine bacterial communities, an in situ time-series experiment was conducted in the oligotrophic coastal waters of the Bermuda Platform. Our goals were to identify polyolefin colonizing taxa and isolate bacterial cultures for future studies of the biochemistry of microbe-plastic interactions. HDPE, LDPE, PP, and glass coupons were incubated in surface seawater for 11 weeks and sampled at two-week intervals. 16S rDNA sequencing and ATR-FTIR/HIM were used to assess biofilm community structure and chemical changes in polymer surfaces. The dominant colonizing taxa were previously reported cosmopolitan colonizers of surfaces in marine environments, which were highly similar among the different plastic types. However, significant differences in rare community composition were observed between plastic types, potentially indicating specific interactions based on surface chemistry. Unexpectedly, a major transition in community composition occurred in all material treatments between days 42 and 56 (p < 0.01). Before the transition, Alteromonadaceae, Marinomonadaceae, Saccharospirillaceae, Vibrionaceae, Thalassospiraceae, and Flavobacteriaceae were the dominant colonizers. Following the transition, the relative abundance of these taxa declined, while Hyphomonadaceae, Rhodobacteraceae and Saprospiraceae increased. Over the course of the incubation, 8,641 colonizing taxa were observed, of which 25 were significantly enriched on specific polyolefins. Seven enriched taxa from families known to include hydrocarbon degraders (Hyphomonadaceae, Parvularculaceae and Rhodobacteraceae) and one n-alkane degrader (Ketobacter sp.). The ASVs that exhibited associations with specific polyolefins are targets of ongoing investigations aimed at retrieving plastic-degrading microbes in culture.

Sulforaphane Bioavailability in Healthy Subjects Fed a Single Serving of Fresh Broccoli Microgreens.

Cruciferous vegetable consumption is associated with numerous health benefits attributed to the phytochemical sulforaphane (SFN) that exerts antioxidant and chemopreventive properties, among other bioactive compounds. Broccoli sprouts, rich in SFN precursor glucoraphanin (GRN), have been investigated in numerous clinical trials. Broccoli microgreens are similarly rich in GRN but have remained largely unexplored. The goal of this study was to examine SFN bioavailability and the microbiome profile in subjects fed a single serving of fresh broccoli microgreens. Eleven subjects participated in a broccoli microgreens feeding study. Broccoli microgreens GRN and SFN contents and stability were measured. Urine and stool SFN metabolite profiles and microbiome composition were examined. Broccoli microgreens had similar GRN content to values previously reported for broccoli sprouts, which was stable over time. Urine SFN metabolite profiles in broccoli microgreens-fed subjects were similar to those reported previously in broccoli sprouts-fed subjects, including the detection of SFN-nitriles. We also reported the detection of SFN metabolites in stool samples for the first time. A single serving of broccoli microgreens did not significantly alter microbiome composition. We showed in this study that broccoli microgreens are a significant source of SFN. Our work provides the foundation for future studies to establish the health benefits of broccoli microgreens consumption.

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.

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.

Age and micronutrient effects on the microbiome in a mouse model of zinc depletion and supplementation.

Older adult populations are at risk for zinc deficiency, which may predispose them to immune dysfunction and age-related chronic inflammation that drives myriad diseases and disorders. Recent work also implicates the gut microbiome in the onset and severity of age-related inflammation, indicating that dietary zinc status and the gut microbiome may interact to impact age-related host immunity. We hypothesize that age-related alterations in the gut microbiome contribute to the demonstrated zinc deficits in host zinc levels and increased inflammation. We tested this hypothesis with a multifactor two-part study design in a C57BL/6 mouse model. The two studies included young (2 month old) and aged (24 month old) mice fed either (1) a zinc adequate or zinc supplemented diet, or (2) a zinc adequate or marginal zinc deficient diet, respectively. Overall microbiome composition did not significantly change with zinc status; beta diversity was driven almost exclusively by age effects. Microbiome differences due to age are evident at all taxonomic levels, with more than half of all taxonomic units significantly different. Furthermore, we found 150 out of 186 genera were significantly different between the two age groups, with Bacteriodes and Parabacteroides being the primary taxa of young and old mice, respectively. These data suggest that modulating individual micronutrient concentrations does not lead to comprehensive microbiome shifts, but rather affects specific components of the gut microbiome. However, a phylogenetic agglomeration technique (ClaaTU) revealed phylogenetic clades that respond to modulation of dietary zinc status and inflammation state in an age-dependent manner. Collectively, these results suggest that a complex interplay exists between host age, gut microbiome composition, and dietary zinc status.

Interplay between Cruciferous Vegetables and the Gut Microbiome: A Multi-Omic Approach.

Brassica vegetables contain a multitude of bioactive compounds that prevent and suppress cancer and promote health. Evidence suggests that the gut microbiome may be essential in the production of these compounds; however, the relationship between specific microbes and the abundance of metabolites produced during cruciferous vegetable digestion are still unclear. We utilized an ex vivo human fecal incubation model with in vitro digested broccoli sprouts (Broc), Brussels sprouts (Brus), a combination of the two vegetables (Combo), or a negative control (NC) to investigate microbial metabolites of cruciferous vegetables. We conducted untargeted metabolomics on the fecal cultures by LC-MS/MS and completed 16S rRNA gene sequencing. We identified 72 microbial genera in our samples, 29 of which were significantly differentially abundant between treatment groups. A total of 4499 metabolomic features were found to be significantly different between treatment groups (q ≤ 0.05, fold change > 2). Chemical enrichment analysis revealed 45 classes of compounds to be significantly enriched by brassicas, including long-chain fatty acids, coumaric acids, and peptides. Multi-block PLS-DA and a filtering method were used to identify microbe−metabolite interactions. We identified 373 metabolites from brassica, which had strong relationships with microbes, such as members of the family Clostridiaceae and genus Intestinibacter, that may be microbially derived.