Dynamic Phaeodactylum tricornutum exometabolites shape surrounding bacterial communities.

Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography-tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual-identified exometabolites. Lastly, we compared responses of a P. tricornutum-adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4-hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4-hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.

Identification of Effector Metabolites Using Exometabolite Profiling of Diverse Microalgae.

Dissolved exometabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of nonpolar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory, freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature-based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of these compounds, five (lumichrome, 5'-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these compounds (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions. IMPORTANCE Microalgae are responsible for nearly half of primary production on earth and play an important role in global biogeochemical cycling as well as in a range of industrial applications. Algal exometabolites are important mediators of algal-algal and algal-bacterial interactions that ultimately affect algal growth and physiology. In this study, we characterize exometabolomes across marine and freshwater algae to gain insights into the diverse metabolites they release into their environments ("exudates"). We observe that while phylogeny can play a role in exometabolome content, environmental conditions or habitat origin (freshwater versus marine) are also important. We also find that several of these compounds can influence algal growth (as measured by chlorophyll production) when provided exogenously, highlighting the importance of characterization of these novel compounds and their role in microalgal ecophysiology.

Agricultural management and plant selection interactively affect rhizosphere microbial community structure and nitrogen cycling.

BACKGROUND: Rhizosphere microbial communities are key regulators of plant performance, yet few studies have assessed the impact of different management approaches on the rhizosphere microbiomes of major crops. Rhizosphere microbial communities are shaped by interactions between agricultural management and host selection processes, but studies often consider these factors individually rather than in combination. We tested the impacts of management (M) and rhizosphere effects (R) on microbial community structure and co-occurrence networks of maize roots collected from long-term conventionally and organically managed maize-tomato agroecosystems. We also explored the interaction between these factors (M × R) and how it impacts rhizosphere microbial diversity and composition, differential abundance, indicator taxa, co-occurrence network structure, and microbial nitrogen-cycling processes. RESULTS: Host selection processes moderate the influence of agricultural management on rhizosphere microbial communities, although bacteria and fungi respond differently to plant selection and agricultural management. We found that plants recruit management-system-specific taxa and shift N-cycling pathways in the rhizosphere, distinguishing this soil compartment from bulk soil. Rhizosphere microbiomes from conventional and organic systems were more similar in diversity and network structure than communities from their respective bulk soils, and community composition was affected by both M and R effects. In contrast, fungal community composition was affected only by management, and network structure only by plant selection. Quantification of six nitrogen-cycling genes (nifH, amoA [bacterial and archaeal], nirK, nrfA, and nosZ) revealed that only nosZ abundance was affected by management and was higher in the organic system. CONCLUSIONS: Plant selection interacts with conventional and organic management practices to shape rhizosphere microbial community composition, co-occurrence patterns, and at least one nitrogen-cycling process. Reframing research priorities to better understand adaptive plant-microbe feedbacks and include roots as a significant moderating influence of management outcomes could help guide plant-oriented strategies to improve productivity and agroecosystem sustainability.

Impacts of Maize Domestication and Breeding on Rhizosphere Microbial Community Recruitment from a Nutrient Depleted Agricultural Soil.

Maize domestication and breeding have resulted in drastic and well documented changes in aboveground traits, but belowground effects on root system functioning and rhizosphere microbial communities remain poorly understood, despite their critical importance for nutrient and water acquisition. We investigated the rhizosphere microbial community composition and structure of ten Zea mays accessions along an evolutionary transect (two teosinte, three inbred maize lines, and five modern maize hybrids) grown in nutrient depleted soil from a low input agricultural system. Microbial community analysis revealed significant differences in community composition between soil compartments (proximal vs. distal rhizosphere) and between plant genetic groups (teosinte, inbred, and modern hybrid). Only a small portion of the microbial community was differentially selected across plant genetic groups: 3.7% of prokaryotic community members and 4.9% of fungal community members were significantly associated with a specific plant genetic group. Indicator species analysis showed the greatest differentiation between modern hybrids and the other two plant genetic groups. Co-occurrence network analysis revealed that microbial co-occurrence patterns of the inbred maize lines' rhizosphere were significantly more similar to those of the teosintes than to the modern hybrids. Our results suggest that advances in hybrid development significantly impacted rhizosphere microbial communities and network assembly.

A New Method to Correct for Habitat Filtering in Microbial Correlation Networks.

Amplicon sequencing of 16S, ITS, and 18S regions of microbial genomes is a commonly used first step toward understanding microbial communities of interest for human health, agriculture, and the environment. Correlation network analysis is an emerging tool for investigating the interactions within these microbial communities. However, when data from different habitats (e.g., sampling sites, host genotype, etc.) are combined into one analysis, habitat filtering (co-occurrence of microbes due to habitat sampled rather than biological interactions) can induce apparent correlations, resulting in a network dominated by habitat effects and masking correlations of biological interest. We developed an algorithm to correct for habitat filtering effects in microbial correlation network analysis in order to reveal the true underlying microbial correlations. This algorithm was tested on simulated data that was constructed to exhibit habitat filtering. Our algorithm significantly improved correlation detection accuracy for these data compared to Spearman and Pearson correlations. We then used our algorithm to analyze a two real data sets of 16S variable region amplicon sequences that were expected to exhibit habitat filtering. Our algorithm was found to effectively reduce habitat effects, enabling the construction of consensus correlation networks from data sets combining multiple related sample habitats.

Metabolomic Analysis Reveals Contributions of Citric and Citramalic Acids to Rare Earth Bioleaching by a Paecilomyces Fungus.

Conventional methods for extracting rare earth elements from monazite ore require high energy inputs and produce environmentally damaging waste streams. Bioleaching offers a potentially more environmentally friendly alternative extraction process. In order to better understand bioleaching mechanisms, we conducted an exo-metabolomic analysis of a previously isolated rare earth bioleaching fungus from the genus Paecilomyces (GenBank accession numbers KM874779 and KM 874781) to identify contributions of compounds exuded by this fungus to bioleaching activity. Exuded compounds were compared under two growth conditions: growth with monazite ore as the only phosphate source, and growth with a soluble phosphate source (K2HPO4) added. Overall metabolite profiling, in combination with glucose consumption and biomass accumulation data, reflected a lag in growth when this organism was grown with only monazite. We analyzed the relationships between metabolite concentrations, rare earth solubilization, and growth conditions, and identified several metabolites potentially associated with bioleaching. Further investigation using laboratory prepared solutions of 17 of these metabolites indicated statistically significant leaching contributions from both citric and citramalic acids. These contributions (16.4 and 15.0 mg/L total rare earths solubilized) accounted for a portion, but not all, of the leaching achieved with direct bioleaching (42 ± 15 mg/L final rare earth concentration). Additionally, citramalic acid released significantly less of the radioactive element thorium than did citric acid (0.25 ± 0.01 mg/L compared to 1.18 ± 0.01 mg/L), suggesting that citramalic acid may have preferable leaching properties for a monazite bioleaching process.

Bioleaching of rare earth elements from monazite sand.

Three fungal strains were found to be capable of bioleaching rare earth elements from monazite, a rare earth phosphate mineral, utilizing the monazite as a phosphate source and releasing rare earth cations into solution. These organisms include one known phosphate solubilizing fungus, Aspergillus niger ATCC 1015, as well as two newly isolated fungi: an Aspergillus terreus strain ML3-1 and a Paecilomyces spp. strain WE3-F. Although monazite also contains the radioactive element Thorium, bioleaching by these fungi preferentially solubilized rare earth elements over Thorium, leaving the Thorium in the solid residual. Adjustments in growth media composition improved bioleaching performance measured as rare earth release. Cell-free spent medium generated during growth of A. terreus strain ML3-1 and Paecilomyces spp. strain WE3-F in the presence of monazite leached rare earths to concentrations 1.7-3.8 times those of HCl solutions of comparable pH, indicating that compounds exogenously released by these organisms contribute substantially to leaching. Organic acids released by the organisms included acetic, citric, gluconic, itaconic, oxalic, and succinic acids. Abiotic leaching with laboratory prepared solutions of these acids was not as effective as bioleaching or leaching with cell-free spent medium at releasing rare earths from monazite, indicating that compounds other than the identified organic acids contribute to leaching performance.

Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.

Metagenomic analysis of a stable trichloroethene-degrading microbial community.

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.

Self-assembly and two-dimensional patterning of cell arrays by electrophoretic deposition.

Using Saccharomyces cerevisiae as a demonstration system, we present a method to form two-dimensional, patternable cellular arrays. The method does not require surface chemical templating of the substratum to produce arrays or patterns. By virtue of their colloidal characteristics, S. cerevisiae cells may be induced to form dense, quasi-ordered two-dimensional clusters adjacent to an electrode surface by electrophoretic deposition (EPD). Using ac EPD, dense two-dimensional cell clusters may be formed in minutes from extremely dilute cell suspensions. The arrays may be induced to form geometric patterns by focusing the electric field during deposition. These monolayer arrays are reversible, dissipating by diffusion on removal of the electric field, and are not in adhesive contact with the electrode surface. Brief application of a modest dc current density adheres the arrays tightly to the surface.