Biology subjects

Gage, D. J.

Publications and source records attributed to Gage, D. J..

10 recordsLinked to original sources

bioRxiv2025

Neuromorpha vorax: a previously unculturable cosmopolitan protist with an unexpectedly complex life-cycle belonging to Glissomonadida Clade-U/Group-TE

Soil protists are increasingly recognized as common members of complex communities that associate with plant root systems, though their contributions to these communities and to the plant host remain obscure. Members Clade-U/Group-TE, are cosmopolitan soil protists and are often among the most abundant protists associated with plant roots. Here we describe the isolation, culturing and characterization of Neuromorpha vorax, a member of the previously uncultured Clade-U/Group-TE branch of the order Glissomonadida. N. vorax grew readily when provided bacteriophage lysate as a food source. This allowed us to grow large numbers of the organisms from single-cell isolates and provided ideal conditions for following transitions from one morphology to another. N. vorax, like most Glissomonads, has a small, flagellated gliding form, but it also displays a wide range of other morphologies including: a crawling form; small and large trophozoites with multiple, long filopodia; small and large resting cysts; clusters of large dividing cells and it displayed cannibalistic feeding behaviors. Given the small size of most Glissomonads, it may be that other members of this important group, known from environmental surveys, but currently uncultured, might also be readily grown on bacteriophage lysates. In addition, given an abundant food source and clear viewing by microscopy, Glissomonads and other small protists may be found to have life cycles and behaviors that are more complex than is currently appreciated. ImportanceProtists from the Clade-U/Group-TE cluster of Glissomonads are wide-spread and abundant colonizers plant roots. Despite being known for over 30 years, they have remained uncultured. We show these protists can be easily cultured using an unusual food source, viral lysates of bacteria. This culturing method allows growth of high numbers of these organisms and reveal thatthey have an unexpectedly complex lifecycle that includes community feeding and cannibalism. Some other, currently unculturable, protists can perhaps be grown with these methods and many of these may also show unexpectedly complex lifecycles. The growth of eukaryotes on virus lysates raises the possibility that viruses in soil may directly contribute to the growth (and not just death) of eukaryotes in soil and root-associated communities.

microbiology↗

bioRxiv2025

Rhizosphere-colonizing bacteria persist in the protist microbiome

Soils contain diverse predatory protists that affect the abundance and behavior of rhizosphere bacteria, including bacteria that may benefit plant health. Protists also harbor their own bacterial microbiomes, including transient and extracellular associates, but it is not known whether the protist microbiome affects the plant rhizosphere. To address this question, we profiled the bacterial microbiomes of eight evolutionarily diverse rhizosphere protist isolates after two years of continuous laboratory culture. We then compared the protist culture microbiomes to maize rhizosphere communities six weeks after protist inoculation. Introduction of protists enriched 13 protist-associated bacterial amplicon sequence variants (ASVs) in the rhizosphere, which comprised [~]10% of the rhizosphere bacterial community. Additional bacterial ASVs ranked highly in abundance in both rhizosphere and protist microbiomes; together, a median 47% of the protist microbiome was enriched or abundant in the rhizosphere. Inoculation with some of the protist cultures positively affected root biomass traits, but a protist mixture had no effect, indicating that the impact of protist holobionts on plant growth is context-dependent. Isolates of protist-associated bacteria had both positive and negative effects on protist growth in culture, suggesting that the bacteria use multiple strategies to survive in proximity to predators. This study demonstrates that even after prolonged laboratory culture, evolutionarily diverse rhizosphere protists host bacterial microbiomes dominated by plant-colonizing bacteria that impact the rhizosphere microbiome after inoculation. The findings suggest that protists may contribute to the rhizosphere as part of the soil microbial seedbank, and identify bacterial groups that may be important to the plant-protist interaction. ImportanceUnderstanding the impact of predatory protists on the plant microbiome will be essential to deploy protists in sustainable agriculture. This study shows that eight rhizosphere protist isolates hosted diverse and distinct bacterial communities, and that a large proportion of these bacteria could be found colonizing the maize root environment long after protists were introduced. This study demonstrates that maize rhizosphere bacteria can persist for years in the protist microbiome, indicating that extremely diverse eukaryotes could help select and maintain rhizosphere bacteria in the absence of the plant.

microbiology↗

bioRxiv2024

Addition of Soil Protists Enhances Performance of Agrochemical Seed Treatments

Chemicals are an integral part of modern agriculture, and are applied through a variety of methods. Some agrochemicals applied for crop protection function by absorption through the root before translocation to the rest of the plant. To be absorbed by the root, the agrochemical must first be transported through the soil, often by water. Some agrochemicals suffer from poor water-based soil transmission due to their chemical properties, limiting their application as a traditional seed treatment. Two such agrochemicals are chlorantraniliprole and spinosad. Soil protists are an important component of the soil microbial community. Certain soil protists have been previously shown to facilitate transport and targeted delivery of suspended particles and cells through soil and microfluidic devices. We provide practical evidence that a soil protist, Colpoda sp., when co-inoculated with an agrochemical seed treatment, can substantially and robustly reduce subsequent pest feeding damage compared with the agrochemical alone. Using maize (Zea mays L.) and fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), in a plant damage assay, we directly compare pest feeding damage and mortality in plants that received no additional treatment, only protists, only agrochemical, and co-inoculation of agrochemical with protists. We discover for both agrochemicals tested, the co-inoculation of protists with the agrochemical increases protection in leaves when the efficacy of the agrochemical alone declines. Protist amendment is a simple, natural, inexpensive, chemical-free, soil-based transport enhancer that thus may be widely useful in a variety of contexts including more sustainable and cost-effective integrated pest management. ImportancePest resistance, regulatory pressure, and environmental concerns are limiting many classes of pesticides which can be effectively used to protect valuable crops from pests. Other classes of pesticides, however, are limited by physical characteristics - water solubility or octane-water partition coefficient (KOW) fall outside the limit for an effective seed coating, or the per-unit cost is high enough to discourage broad application. Here, we provide data which supports the co-inoculation of a high value, low solubility, high KOW pesticides with a naturally-occurring soil protist as a seed treatment can enhance crop protection relative to the pesticide alone. This co-formulation reduced feeding damage by up to 30% compared with the pesticide alone. Co-inoculation of crop-protecting agrochemicals with natural soil protists may be employed as a more sustainable agriculture biotechnology, enabling the use of classes of agrochemicals which may not otherwise show sufficient performance for use as a seed treatment.

microbiology↗

bioRxiv2021

Soil protists can actively redistribute beneficial bacteria along Medicago truncatula roots

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria, all of which play a significant role in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a usable form of nitrogen for plants. S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system who prey on soil bacteria and have been known to egest undigested phagosomes. We show that the soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. By using pseudo-3D soil microcosms, we directly observed the presence of fluorescently labelled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal was detected 52 mm, on average, farther down the roots when Colpoda sp. was also present compared with the experimental treatment that contained bacteria but not protists. Direct counts also showed that protists are required for viable bacteria to reach the deeper sections of root systems in our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. As a sustainable agriculture biotechnology, protist-facilitated transport has the potential to boost efficacy of bacterial inoculants, thereby helping growers avoid overuse of nitrogen fertilizers and enhance performance of climate-smart, no-till farming practices. ImportanceSoil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

microbiology↗

bioRxiv2021

A dual sequence and culture-based survey of maize rhizosphere protists reveals dominant,plant-enriched, and culturable community members

Protists play important roles in shaping the microbial community of the rhizosphere. However, there is still a limited understanding of how plants shape the protist community, and how well protist isolate collections might represent rhizosphere protist composition and function in downstream studies. We sought to determine whether maize roots select for a distinct protist community in the field, and whether the common or dominant members of that community are readily culturable using standard protist isolation methods. We sequenced 18S and 16S rRNA genes from the rhizospheres of maize grown in two sites, and isolated 103 protists into culture from the same roots. While field site had the greatest effect, rhizospheres in both sites had distinct protist composition from the bulk soils, and certain taxa were enriched in both sites. Enriched taxa were correlated to bacterial abundance patterns. The isolated protists represented six supergroups, and the majority corresponded to taxa found in the sequencing survey. Twenty-six isolates matched eight of the 89 core rhizosphere taxa. This study demonstrates that maize roots select for a distinct protist community, but also illustrate the potential challenges in understanding the function of the dominant protist groups in the rhizosphere. Originality-Significance StatementThis is the first study comparing cultivation-dependent and independent methods for studying the protist community of plant roots, and the first untargeted analysis of the maize rhizospheres effect on protist communities. We show that maize in different sites select for distinct communities and overlapping enriched taxa, but that isolating the most important plant-associated protists may be a challenge for researchers.

microbiology↗

bioRxiv2020

Simultaneous Single-Cell Genome and Transcriptome Sequencing of Termite Hindgut Protists Reveals Metabolic and Evolutionary Traits of Their Endosymbionts

Different protist species which colonize the hindguts of wood feeding Reticulitermes termites are associated with endosymbiotic bacteria belonging to the genus Endomicrobium. In this study, we focused on the endosymbionts of three protist species from Reticulitermes flavipes, which included Pyrsonympha vertens, Trichonympha agilis, and Dinenympha species II. Since these protist hosts represented members of difference taxa which colonize different niches within the hindguts of their termite hosts, we investigated if these differences translated to differential gene content and expression in their endosymbionts. Following assembly and comparative genome and transcriptome analyses, we discovered that these endosymbionts differed with respect to possible niche specific traits such carbon metabolism. Our analyses supported that genes related to carbon metabolism were acquired by horizontal gene transfer (HGT) from donor taxa which are present in termites hindgut community. In addition, our analyses supported that these endosymbionts have retained and expressed genes related to natural transformation (competence) and recombination. Taken together, the presence of genes acquired by HGT and a putative competence pathway supported that these endosymbionts are not cut-off from gene flow and that competence may be a mechanism by which members of the Endomicrobium can acquire new traits. ImportanceThe composition and structure of wood, which contains cellulose, hemicellulose and lignin, prevents most organisms from using this common food source. Termites are a rare exception among animals, and they rely on a complex microbiome housed in their hindguts to use wood as a source of food. The lower termite R. flavipes houses a variety of protist and prokaryotes that are the key players the disassembly of lignocellulose. In this paper we describe the genomes and the gene expression profiles of five Endomicrobium endosymbionts living inside three different protist species from R. flavipes. Data from these genomes suggest that these Endomicrobium species have different mechanisms for using both carbon and nitrogen. In addition, they harbor genes that may be used to import free DNA from their environment. This process of DNA-uptake may cntribute to the high levels of horizontal gene transfer often seen in the Endomicrobium species.

microbiology↗

bioRxiv2020

Development and application of aerobic, chemically defined media for Dysgonomonas

Members of Dysgonomonas are Gram-negative, non-motile, facultatively anaerobic coccobacilli originally described in relation to their isolation stool and wounds of human patients (CDC group DF-3). More recently Dysgonomonas have been found to be widely distributed in terrestrial environments and are particularly enriched in insect systems. Their prevalence in xylophagous insects such as termites and wood-feeding cockroaches, as well as in soil-fed microbial fuel cells, elicit interest in lignocellulose degradation and biofuel production, respectively. Their prevalence in mosquito and fruit fly have implications relating to symbiosis, host immunology and developmental biology. Additionally, their prevalence in termite, mosquito and nematode present novel opportunities for pest and vector control. Currently, the absolute growth requirements of Dysgonomonas are unknown, and they are cultured solely under anaerobic conditions on complex media containing blood, peptones, tryptones, and yeast, plant or meat extracts. Restrictive & undefined culturing conditions preclude physiological and genetic studies, and thus further understanding of metabolic potential. Here we describe the requirements for growth of termite-derived Dysgonomonas isolates and create parallel defined, minimal and complex media that permit vigorous and reliable aerobic growth. Furthermore, we show that these media can be used to easily enrich for Dysgonomonas isolates from complex and microbially-diverse environmental samples. Impact StatementMembers of the genus Dysgonomonas are increasingly prevalent in ecological, medical and biotechnological contexts. To the best of our knowledge, there are currently no formulations for chemically defined or minimal media for Dysgonomonas, and limited complex formulations that allow aerobic growth, particularly on solid media. We have created three parallel media formulations (complex, defined & minimal) that permit robust aerobic and anaerobic growth in liquid and agar-solidified media. These formulations remove the necessity for animal blood and expensive equipment such as anaerobic chambers, which can inhibit basic research by groups with biosafety and resource limitations.

microbiology↗

bioRxiv2020

Optogenetics in Sinorhizobium meliloti enables spatial control of exopolysaccharide production and biofilm structure

Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Due to the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under non-inducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ. SignificanceMicroorganisms are key players in sustaining the soil environment and plant growth. Symbiotic associations of soil microbes and plants provide a major source of nitrogen in agricultural systems, prevent water contamination from synthetic fertilizer application, and support crop growth in marginal soils. However, measuring the impact of microbial gene products on beneficial function remains a major challenge. This work provides a critical step toward addressing this challenge by implementing external gene regulation in a well characterized nitrogen fixing soil bacterium. We show that light exposure enables spatial and temporal control of the extracellular polysaccharide production functionality essential for symbiosis. Remote control of genes enables the benefits of candidate microorganisms to be systematically measured and enhanced within complex natural settings.

synthetic biology↗

bioRxiv2020

Validation of a PNA clamping method for reducing host DNA amplification and increasing eukaryotic diversity in rhizosphere microbiome studies

Protists and microscopic animals are important but poorly understood determinants of plant health. Plant-associated eukaryotes could be surveyed by high-throughput sequencing of 18S ribosomal RNA (rRNA) genes, but the abundance of plant DNA in rhizosphere samples makes 18S rRNA gene amplification with universal primers unfeasible. Here we applied a pipeline to generate peptide nucleic acid (PNA) clamps to suppress the amplification of plant host DNA during 18S rRNA gene library preparation. We designed a PNA clamp, PoacV9_01, specific to the V9 region of the 18S rRNA gene for plants in the family Poaceae. PoacV9_01 suppressed the amplification of five species of grain crops in quantitative PCR reactions. In an 18S rRNA gene sequencing survey of the rhizosphere of maize, PoacV9_01 reduced the relative abundance of plant reads from 65% to 0.6%, while drastically increasing the number and diversity of animal, fungal, and protist reads detected. Thus, PoacV9_01 can be used to facilitate the study of eukaryotes present in grass phytobiomes. In addition, the pipeline developed here can be used to develop PNA clamps that target other plant species.

microbiology↗

bioRxiv2018

Oligotyping Bacterial Symbionts of Hindgut Protists Reveals Complex Population Structures and Transmission Trends in the Termite Reticulitermes flavipes

The hindgut protists of wood-feeding termites are usually colonized by prokaryotic symbionts. Many of the hurdles that have prevented a better understanding of these symbionts arise from variation among protist and termite host species and the inability to maintain prominent community members in culture. These issues have made it difficult to study the fidelity, acquisition, and differences in colonization of protists by bacterial symbionts. In this study, we use high throughput amplicon sequencing of the V4 region of 16S rRNA genes to determine the composition of bacterial communities associated with single protist cells of six protist species, from the genera Pyrsonympha, Dinenympha, and Trichonympha that are present in the hindgut of the termite Reticulitermes flavipes. By analyzing amplicon sequence variants (ASVs), the diversity and distribution of protist-associated bacteria was compared within and across these six different protist species. ASV analysis showed that, in general, each protist genus associated with a distinct community of bacterial symbionts which were conserved across different termite colonies. However, some ASVs corresponding to ectosymbionts (Spirochaetes) were shared between different Dinenympha species and to a lesser extent with Pyrsonympha and Trichonympha hosts. This suggested that certain bacterial symbionts may be cosmopolitan to some degree and perhaps acquired by horizontal transmission. Using a fluorescence-based cell assay, we could observe the horizontal acquisition of surface-bound bacteria. This acquisition was shown to be time-dependent, involve active processes, and was non-random with respect to binding locations on some protists.

microbiology↗