Results for “microbial community” — Page 37

DOE OSTI2023

Root and Microbial Soil CO 2 and CH 4 Fluxes Respond Differently to Seasonal and Episodic Environmental Changes in a Temperate Forest

Upland forest soils are typically major atmospheric carbon dioxide (CO 2 ) sources and methane (CH 4 ) sinks, but the contributions of root and microbial processes, as well as their separate temporal responses to environmental change, remain poorly understood. This 2-year study was conducted in a temperate, deciduous forest located on the Chesapeake Bay in Maryland, USA. We used temporal CO 2 and CH 4 flux measurements, exclusion-source partitioning, and an ecosystem-scale flooding experiment to understand how carbon (C) fluxes, and their root and microbial sources, respond to seasonal and episodic environmental change. Here, we show that the root-and-rhizosphere component of soil CO 2 and CH 4 flux is significant and that its dependence on soil temperature and volumetric water content (VWC) influences soil C dynamics at seasonal timescales. Experimental flooding shows that CO 2 and CH 4 flux responses to episodic moisture change were driven by suppression of soil heterotrophs, while root respiration did not respond to transient hydrologic disturbance. Methane uptake responded strongly to episodic inundation, reinforcing the important role of soil moisture in the short-term control of the forest soil CH 4 sink. However, despite the clear seasonality of CH 4 uptake, as well as its strong response to short-term experimental inundation, temperature and VWC were weak predictors of CH 4 uptake at a seasonal timescale. We suggest that CH 4 consumption in the long-term may be determined by vegetation, nutrients, microbial communities, or other factors correlated with seasonal changes. Our results indicate that root and microbial sources of both CO 2 and CH 4 flux respond differently in timing and magnitude to seasonal and episodic environmental change.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Soil microbiome engineering for sustainability in a changing environment

Recent advances in microbial ecology and synthetic biology have the potential to mitigate damage caused by anthropogenic activities that are deleteriously impacting Earth’s soil ecosystems. Here, we discuss challenges and opportunities for harnessing natural and synthetic soil microbial communities, focusing on plant growth promotion under different scenarios. We explore current needs for microbial solutions in soil ecosystems, how these solutions are being developed and applied, and the potential for new biotechnology breakthroughs to tailor and target microbial products for specific applications. We highlight several scientific and technological advances in soil microbiome engineering, including characterization of microbes that impact soil ecosystems, directing how microbes assemble to interact in soil environments, and the developing suite of gene-engineering approaches. This Review underscores the need for an interdisciplinary approach to understand the composition, dynamics and deployment of beneficial soil microbiomes to drive efforts to mitigate or reverse environmental damage by restoring and protecting healthy soil ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Who Is Metabolizing What? Discovering Novel Biomolecules in the Microbiome and the Organisms Who Make Them

Even as the field of microbiome research has made huge strides in mapping microbial community composition in a variety of environments and organisms, explaining the phenotypic influences on the host by microbial taxa – both known and unknown – and their specific functions still remain major challenges. A pressing need is the ability to assign specific functions in terms of enzymes and small molecules to specific taxa or groups of taxa in the community. This knowledge will be crucial for advancing personalized therapies based on the targeted modulation of microbes or metabolites that have predictable outcomes to benefit the human host. This perspective article advocates for the combined use of standards-free metabolomics and activity-based protein profiling strategies to address this gap in functional knowledge in microbiome research via the identification of novel biomolecules and the attribution of their production to specific microbial taxa.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

The microbiologist's guide to metaproteomics

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. By integrating metaproteomics with other omics disciplines, researchers can gain a comprehensive understanding of microbial ecology, interactions, and functional dynamics. This review, developed by the Metaproteomics Initiative (www.metaproteomics.org), serves as a practical guide for both microbiome and proteomics researchers, presenting key principles, state-of-the-art methodologies, and analytical workflows essential to metaproteomics. Topics covered include experimental design, sample preparation, mass spectrometry techniques, data analysis strategies, and statistical approaches.

bioinformatics↗

DOE OSTI2022

Soil viruses: Understudied agents of soil ecology

Over the past couple decades there has been intense exploration of the soil microbiome using modern sequencing and bioinformatics approaches. These studies have revealed a vast diversity of microorganisms across a variety of habitats (Thompson et al., 2017) and how differences in the environment, for example with climate change, influence the soil microbial community composition (Jansson and Hofmockel, 2020). The focus of these studies has primarily been on microbial cellular organisms, including bacteria, archaea and fungi. By contrast, in depth study of soil viruses has been largely neglected, until recently. There are several reasons that soil viruses are less studied, including difficulties with extraction of viruses from soil particles, difficulties with classification of soil viruses based on microscopic imaging, and reliance on the small proportion of microbial hosts that can be cultivated. Now that comprehensive soil metagenomes are available the current trend has been to survey the metagenomes for viral sequences. This approach has revealed that soil viruses are incredibly abundant, highly diverse, and largely uncharacterized (Paez-Espino et al., 2016). This so-called ‘viral dark matter’ presents an incredible research opportunity for the future. Here I will highlight some of these potential research avenues based on what is known to date.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Microbial metabolic dependency and its impacts on the soil carbon cycle (Final Technical Report, DE-SC0016364)

The terrestrial biosphere contains a large fraction of global carbon (C) and nearly 70% of the organic C in terrestrial systems is found in soils. Global changes in atmospheric CO 2 , temperature, precipitation, and ecosystem N inputs, will impact primary production and carbon inputs to soils, but it remains difficult to predict soil responses to these environmental conditions. Our ability to predict C-cycle responses to global change remains limited by unexplained variability within the terrestrial C-cycle. Our difficulty in predicting responses of the soil C-cycle to environmental change suggests a need for a greater understanding of the biotic mechanisms that govern soil C-transformations. Changes in microbial community structure and function have been proposed to impact soil processes, but mechanistic connections between microbial activity and C-cycle transformations remain poorly described. We assembled an interdisciplinary team to conduct cross-scale examination of microbial contributions to C-cycle dynamics and C-fate in soils. We investigated microbial contributions to soil C-transformations using a suite of complementary isotopic techniques including DNA-Stable Isotope Probing (DNA-SIP), metagenomic-SIP, metabolomic-SIP, proteomic-SIP, and microspatial analysis of soil organic C. These techniques were deployed in experiments that tested fundamental assumptions that underlie terrestrial C-cycle models. We characterized the functional characteristics of soil microorganisms responsible for major C-transformations in soil, evaluated the metabolic and ecological interactions that underlie soil C-cycle dynamics, and evaluated the degree to which these interactions impact rates of C mineralization and stabilization in soils.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

From wolves to humans: oral microbiome resistance to transfer across mammalian hosts

The mammalian mouth is colonized by complex microbial communities, adapted to specific niches, and in homeostasis with the host. Individual microbes interact metabolically and rely primarily on nutrients provided by the host, with which they have potentially co-evolved along the mammalian lineages. The oral environment is similar across mammals, but the diversity, specificity, and evolution of community structure in related or interacting mammals are little understood. Here, we compared the oral microbiomes of dogs with those of wild wolves and humans. In dogs, we found an increased microbial diversity relative to wolves, possibly related to the transition to omnivorous nutrition following domestication. This includes a larger diversity of Patescibacteria than previously reported in any other oral microbiota. The oral microbes are most distinct at bacterial species or strain levels, with few if any shared between humans and canids, while the close evolutionary relationship between wolves and dogs is reflected by numerous shared taxa. More taxa are shared at higher taxonomic levels including with humans, supporting their more ancestral common mammalian colonization followed by diversification. Phylogenies of selected oral bacterial lineages do not support stable human-dog microbial transfers but suggest diversification along mammalian lineages (apes and canids). Therefore, despite millennia of cohabitation and close interaction, the host and its native community controls and limits the assimilation of new microbes, even if closely related. Higher resolution metagenomic and microbial physiological studies, covering a larger mammalian diversity, should help understand how oral communities assemble, adapt, and interact with their hosts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Genetic modification of the shikimate pathway to reduce lignin content in switchgrass ( Panicum virgatum L.) significantly impacts plant microbiomes

Switchgrass (Panicum virgatum L.) is considered a sustainable biofuel feedstock, given its fast-impact growth, low input requirements, and high biomass yields. Improvements in bioenergy conversion efficiency of switchgrass could be made by reducing its lignin content. Engineered switchgrass that expresses a bacterial 3-dehydroshikimate dehydratase (QsuB) has reduced lignin content and improved biomass saccharification due to the rerouting of the shikimate pathway towards the simple aromatic protocatechuate at the expense of lignin biosynthesis. However, the impacts of this QsuB trait on switchgrass microbiome structure and function remain unclear. To address this, wild-type and QsuB-engineered switchgrass were grown in switchgrass field soils, and samples were collected from inflorescences, leaves, roots, rhizospheres, and bulk soils for microbiome analysis. We investigated how QsuB expression influenced switchgrass-associated fungal and bacterial communities using high-throughput Illumina MiSeq amplicon sequencing of ITS and 16S rDNA. Compared to wild-type, QsuB-engineered switchgrass hosted different microbial communities in roots, rhizosphere, and leaves. Specifically, QsuB-engineered plants had a lower relative abundance of arbuscular mycorrhizal fungi (AMF). Additionally, QsuB-engineered plants had fewer Actinobacteriota in root and rhizosphere samples. These findings may indicate that changes in the plant metabolism impact both AMF and Actinobacteriota similarly or potential interactions between AMF and the bacterial community. This study enhances understanding of plant-microbiome interactions by providing baseline microbial data for developing beneficial bioengineering strategies and by assessing nontarget impacts of engineered plant traits on the plant microbiome.

09 BIOMASS FUELS↗

DOE OSTI2023

Dredged sediments contain potentially beneficial microorganisms for agriculture and little harmful cyanobacteria

Soils worldwide are degrading, raising concerns about our ability to feed the growing global population. Soil amendments that can alleviate degradation are gaining attention. The application of sediments dredged from waterways to agricultural fields has increasing promise as a means for improving degraded soils. However, herbaceous plant species may have difficulty establishing on dredged material because of low nutrient availability, inhibitory levels of toxins, unsuitable moisture conditions and lack of microorganisms capable of ameliorating these characteristics. To counteract these issues, we sought to understand if the use of a cover crop would increase the abundance, diversity and function of beneficial soil microorganisms compared to harmful microorganisms in dredged sediments. We collected soil samples from two 100% dredged sediment plots, one where winter cereal rye (Secale cereal) was grown as a winter cover crop and one left fallow over the winter, followed by traditional corn (Zea mays) planting. We sampled both plots three times during the growing season: before cover crop application, following cover crop application but before corn planting and following final corn harvest. We then used high-throughput sequencing to identify the bacterial and fungal communities present in the samples. Our data show that cover crop application did not alter the microbial community in these plots. However, sampling time decreased species diversity and altered the composition of both fungal and bacterial communities recovered from these plots. Across both plots, microorganisms associated with carbon cycling were more abundant than those associated with harmful effects, including microcystin-producing cyanobacteria, which were an extremely small portion of the overall community. Our work suggests that dredged sediments have the potential to improve soil function through the addition of microorganisms associated with nutrient cycling, but a cover crop is not necessary to incur these benefits.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2004

Astrobiology of Comets

We review the current state of knowledge concerning microbial extremophiles and comets and the potential significance of comets to Astrobiology. We model the thermal history of a cometary body, regarded as an assemblage of boulders, dust, ices and organics, as it approaches a perihelion distance of - IAU. The transfer of incident energy from sunlight into the interior leads to the melting of near surface ices, some under stable porous crust, providing possible habitats for a wide range of microorganisms. We provide data concerning new evidence for indigenous microfossils in CI meteorites, which may be the remains of extinct cometary cores. We discuss the dominant microbial communities of polar sea-ice, Antarctic ice sheet, and cryoconite environments as possible analogs for microbial ecosystems that may grow in sub-crustal pools or in ice/water films in comets.

Hoover, Richard B.↗

NASA NTRS2017

Microbial Characteristics of ISS Environmental Surfaces

The microbiome of environmental surfaces from the International Space Station were characterized in order to examine the relationship to crew and hardware maintenance. The Microbial Observatory (ISS-MO) experiment generated a microbial census of ISS environments using advanced molecular microbial community analyses along with traditional culture-based methods. Since the “omics” methodologies generated an extensive microbial census, significant insights into spaceflight-induced changes in the populations of beneficial and/or potentially harmful microbes were gained. Surface samples were collected from several ISS surface locations from three flight opportunities, and were returned to Earth via the Soyuz TMA-14M or the Space X Dragon capsule. In addition to cultivation methods, viable microbial burden, iTag-based sequencing, and metagenome analyses were carried out. The cultivable microbial bioburden differed by location and sampling event. Exploring the ISS environmental microbiome revealed presence of opportunistic pathogens and antibiotic resistant microbes. Genes involved in ATP binding cassette transporters, two component systems, and beta-lactam resistance were among a diverse set of metabolic and genetic information processing pathways. Whole genome sequencing (WGS) of 50 ISS strains exhibiting resistance to various antibiotics was carried out. The antibiotic resistant genes deduced from the WGS were compared with the resistomes generated directly from the gene pool of the environmental samples. Two unique Aspergillus fumigatus strains isolated from the ISS were characterized and compared to the experimentally established clinical isolates Af293 and CEA10. A virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis indicated that both ISSFT-021 and IF1SW-F4 were significantly more lethal compared to Af293 and CEA10. The findings from this Environmental “Omics” project should be exploited to enhance human health and well-being of a closed system. In other words, the ISS-MO research aims to "translate" findings in fundamental research into medical practice (pathogen detection) and meaningful health outcomes (countermeasure development).

Perry, Jay↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

NASA NTRSDate not supplied

ISS External Microorganisms: Tools and Techniques for Collecting Planetary Protection Samples During Extravehicular Activity

We have designed, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars.

Planetary Protection↗

NASA NTRSDate not supplied

ISS External Microorganisms: Collecting Planetary Protection Samples During Extravehicular Activity

Introduction: We have designed1, built, and tested a sampling kit (Fig. 1) to aseptically collect microbiological samples from exterior surfaces on the ISS (International Space Station). The kit was flown to ISS as part of the NG-19 commercial cargo mission in August of 2023. Astronauts will use the kit to collect samples from six exterior surfaces on ISS. These samples will be frozen at -80°C after collection and returned to the ground for next generation DNA sequencing. The results of this experiment will help inform planetary protection requirements for crewed missions. We hypothesize that there are detectable microbial communities outside ISS and that these communities originate from inside ISS. Current life support systems do not include any components for reducing microbial leakage. Gases are vented from inside ISS without filtration and there are currently no protocols in place to minimize bioburden on space suit exteriors prior to use. It is important to quantify the bioburden on current vehicles so that achievable limits can be set for crewed missions to astrobiologically relevant locations like Mars. Figure 1: The sampling kit contains eight swab canisters in total (six on the top and two on the bottom). A resuable end effector (1) is used to remove and reinstall the swabs. The kit also contains a handrail (2), tether loop (3), and, bayonet probes(4) to allow easy manipulation during EVA. Methods: We have designed a kit to carry 8 commercially available foam swabs into and out of vacuum without compromising the swabs’ sterility. The specially designed swab canisters contain a 0.2 µm Teflon filter that allows the canister to accommodate pressure changes without introducing unwanted contaminants. The kit meets existing EVA (Extravehicular Activity) safety requirements and has been used by human test subjects in the neutral buoyance lab and at vacuum in test chambers at the Johnson Space Center. In the early part of 2024, astronauts will use this sampling kit to swab six surfaces on the exterior of ISS. We will collect samples from: the airlock vestibule, the interior surface of the airlock thermal cover, an exterior handrail, a vent connected to the CO2 removal system, and a vent connected to the payload vacuum system inside ISS. Approximately 300 cm2 will be swabbed at each location. A seventh swab will be exposed to the vacuum of space without touching any surfaces as a blank. The eighth swab will remain sealed as a process control. After the EVA the swab kit will be frozen at -80°C and returned to earth at the earliest possible opportunity. The samples will remain frozen until they are thawed for next generation DNA sequencing on earth. We will use amplicon sequencing to identify any bacteria, archaea or fungi present in the samples. If there is enough DNA present, we will use shotgun metagenomic sequencing to further characterize the microbial ecology outside ISS. Preliminary Results: Results from ground-based testing demonstrate that the swab kit is capable of cycling in and out of vacuum without contaminating the swabs. Test subjects, wearing flight-like EVA gloves could remove swabs from the canister and sample discrete locations without inadvertently touching any other surfaces. Several types of bacteria and fungi were collected during these ground tests and survived up to 6 hours at vacuum. This includes non-spore forming bacterial like Staphyloccus capitis that are not traditionally considered extremophiles. Shotgun metagenomic sequencing of the ground-test samples revealed bacteria associated with human skin and airways on the exterior of space suits used during these tests 2 In addition to these results we will present preliminary results from our space-flight samples. We will also present lessons learned from attempting to collect microbiological samples during an EVA and describe how our results will affect planetary protection requirements for future crewed missions.

Aaron B. Regberg↗

NASA NTRS1988

Search for Martian fossil communities: Science strategies, sediment sites, and sample handling

The strategy for locating and sampling possible fossilized Martian organisms benefits from our experience with fossil microbial ecosystems on Earth. Evidence of early life is typically preserved as stromatolites in carbonates and cherts, and as microfossils in cherts, carbonates and shales. Stromatolites, which are laminated flat or domal structures built by microbial communities, are very likely the oldest and most widespread relics of early life. These communities flourished in supratidal to subtidal coastal benthic environments, wherever sunlight was available and where incoming sediments were insufficient to bury the communities before they became established. A logical site for such communities on Mars might be those areas in an ancient lake bed which were furthest from sediment input, but were still sufficiently shallow to have received sunlight. Therefore, although some sites within Valles Marineris might have contained ponded water, the possibly abundant sediment inputs might have overwhelmed stromatolite-like communities. Localized depressions which acted as catchment basins for ancient branched valley systems might be superior sites. Perhaps such depressions received drainage which, because of the relatively modest water discharges implied for these streams, was relatively low in transported sediment. Multiple streams converging on a single basin might have been able to maintain a shallow water environment for extended periods of time.

Desmarais, David J.↗

DOE OSTI2021

Manganese uptake by red maples in response to mineral dissolution rates in soil

Manganese (Mn) is an essential plant micronutrient that influences photosynthesis, ecosystem productivity, and soil carbon storage. Our objective was to quantify how Mn uptake by forest vegetation relates to Mn release into soil solution through mineral dissolution. In this work, a greenhouse pot experiment was conducted to quantify Mn uptake by red maple saplings as a function of mineral solubility to test whether Mn uptake was limited by the supply of Mn to soil solution. Differences in soil microbial community composition between treatments, particularly amongst Mn cycling bacteria and fungi, were also evaluated to assess potential microbial impacts on observed Mn fluxes. Plant Mn uptake was highest in systems supplied with dissolved Mn(II) because it was not kinetically limited by mineral weathering. Mn uptake was also higher in systems supplied with a fast-weathering substrate (shale containing Mn(II)-bearing pyrite) than a slow-weathering substrate (Mn(IV)-oxide). However, vegetation accumulated a decreasing proportion of available Mn with increasing solubility, indicating that uptake was tempered relative to solubility. The presence of bacterial phyla containing known Mn-oxidizing bacteria indicates the potential for Mn-oxidizing bacteria to influence Mn solubility within Mn-oxide and dissolved Mn treatments. A relatively low abundance of these bacteria points to their limited capacity to oxidize and retain Mn in the shale treatments, consistent with substantial Mn leaching. We conclude that Mn uptake was primarily controlled by dissolution rates of Mn-bearing minerals but modified by biological processes. The quantitative framework presented here can guide understanding of how biogeochemical processes control element cycling between plants and soils.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Plant root exudates and rhizosphere bacterial communities shift with neighbor context

A plant's neighborhood context can alter its interactions with other organisms, but little is known about how these dynamics occur belowground, especially with soil microbes. Microbial communities in rhizosphere soil are influenced by many factors, including abiotic conditions and root-derived signals. In particular, root exudates have strong effects on rhizosphere assembly, respond to changes in abiotic conditions, and help plants interact with neighbors. Therefore, we predicted that root exudates likely play a central role in neighbor-induced shifts in rhizosphere communities. We conducted a greenhouse experiment to test this and determine how the rhizosphere bacterial community of a focal plant, Panicum virgatum, changed when beside different neighbors, and whether these shifts were mediated by neighbor-induced changes in root exudation. We found that neighbor altered both focal plant exudates and rhizosphere community, and that changes were largest when the focal plant was beside the most competitive neighbor, Rudbeckia hirta, which reduced both focal plant growth and nitrogen uptake. Several factors contributed to neighbor impacts on rhizosphere assembly, including neighbor-induced changes in root exudates during nitrogen-limitation and microbial spillover from roots of larger neighbors. Using an additional soil incubation, we also found that these changes in exudates can have even greater effects on soil nutrients than on microbial assembly. Overall, we show that neighbors influence one another's microbiomes, and highlight neighbor-induced changes in root exudates as one mechanism through which this may occur. Furthermore, this work suggests that rhizosphere assembly may differ in mixed-species communities and thus emphasizes a need for microbiome studies that consider neighborhood context.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infectEscherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targetedE. colicells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.

Science & Technology - Other Topics↗