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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays. Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant-microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Yadav, Archana↗
DOE OSTI2023

Plant myo -inositol transport influences bacterial colonization phenotypes

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in seedlings of the model plant Arabidopsis thaliana to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound has been linked to increased host colonization, we identify bacterial phenotypes that occur in both catabolism-dependent and -independent manners, suggesting that myo-inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggest host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite myo-inositol.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Geographic patterns and determinants of antibiotic resistomes in coastal sediments across complex ecological gradients

Coastal areas are highly influenced by terrestrial runoffs and anthropogenic disturbances, commonly leading to ecological gradients from bay, nearshore, to offshore areas. Although the occurrence and distribution of sediment antibiotic resistome are explored in various coastal environments, little information is available regarding geographic patterns and determinants of coastal sediment antibiotic resistomes across ecological gradients at the regional scale. Here, using high-throughput quantitative PCR, we investigated the geographic patterns of 285 antibiotic resistance genes (ARGs) in coastal sediments across a ~ 200 km scale in the East China Sea. Sediment bacterial communities and physicochemical properties were characterized to identify the determinants of sediments antibiotic resistome. Higher richness and abundance of ARGs were detected in the bay samples compared with those in nearshore and offshore samples, and significant negative correlations between the richness and/or abundance of ARGs and the distance to coastline (DTC) were identified, whereas different types of ARGs showed inconsistency in their relationships with DTC. The composition of antibiotic resistome showed significant correlations with nutrition-related variables (including NH 4 + -N, NO 3 − -N, and total phosphorus) and metals/metalloid (including As, Cu, Ni, and Zn), suggesting that terrestrial disturbances largely shape the antibiotic resistome. The Bipartite network showed strong associations between ARGs and mobile genetic elements (MGEs), and Partial Least Squares Path Modeling further revealed that terrestrial disturbance strength (as indicated by DTC) directly affected abiotic environmental conditions and bacterial community composition, and indirectly affected antibiotic resistome via MGEs. These findings provide insights into regional variability of sediment antibiotic resistome and its shaping path across complex ecological gradients, highlighting terrestrial disturbances as determinative forces in shaping coastal sediment antibiotic resistomes.

Xiong, Shangling↗
DOE OSTI2023

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13 C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Microbiota functional activity biosensors for characterizing nutrient metabolism in vivo

Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was isolated from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionated pea fiber and sugar beet arabinan, on a human gut bacterial strain consortium in gnotobiotic mice. We produced ‘Microbiota Functional Activity Biosensors’ (MFABs) consisting of glycans covalently linked to the surface of fluorescent paramagnetic microscopic glass beads. Three MFABs, each containing a unique glycan/fluorophore combination, were simultaneously orally gavaged into gnotobiotic mice, recovered from their intestines, and analyzed to directly quantify bacterial metabolism of structurally distinct arabinans in different human diet contexts. Colocalizing pea-fiber arabinan and another polysaccharide (glucomannan) on the bead surface enhanced in vivo degradation of glucomannan. MFABs represent a potentially versatile platform for developing new prebiotics and more nutritious foods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetic Fusion of Thermoresponsive Polypeptides with UCST‐type Behavior Mediates 1D Assembly of Coiled‐Coil Bundlemers

Abstract Thermoresponsive resilin‐like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil‐forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature‐triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet‐visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic‐transmission electron microscopy and coarse‐grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

Patkar, Sai S.↗
DOE OSTI2021

19-LW-045 Full Length Final Report. Molecular Mechanisms of Bacterial Pathogenesis: Waging the Arms Race with Superbugs

As the current global pandemic makes abundantly clear, we need a better understanding of infectious disease to safeguard human health, the economy and global security. Modern omics techniques hold the promise of providing a comprehensive understanding of the molecular mechanisms of life, including causes of pathogenesis from infectious disease at the molecular level, but we there is a serious gap in annotation of gene function. For as much as half of the genes and gene products encoded in genomes the molecular and/or cellular function is unknown or only partially understood. Recent innovations in fluorescence microscopy for live cell imaging and genetic engineering make it possible to determine the temporal correlation between molecular events, such as a gene being expressed due to host-pathogen interaction, and cellular events, such as bacterial invasion of immune cells. This is turn allows us to gain new insight as to the molecular and cellular role of individual genes and will enable the discovery and validation of new molecular mechanisms essential for infectious disease. Knowing the molecular mechanisms of disease processes will provide new therapeutic targets or novel countermeasure strategies. We aimed to develop a lattice light sheet fluorescence microscope as a unique resource at LLNL for long time course live cell imaging experiments; to develop the reagents and cell lines needed to monitor molecular events during the course pathogenic bacteria infecting mammalian immune cells; and to demonstrate that we could capture molecular events during an infection. We fully commissioned the LLNL lattice light sheet microscope and conducted initial proof of principle imaging experiments on mammalian immune cells and pathogenic bacteria. It is clear from the experience gained that long time course live cell imaging has tremendous potential to help elucidate molecular mechanisms of host-pathogen interactions and to help annotate gene function, which would establish a basis for new countermeasures. It is also clear that if live cell imaging is to realize its full potential new data processing and analysis tools will need to be developed to facilitate analysis of molecular events within cells; new sample chambers and stages could facilitate studies with a wider range of cell and tissue types; and alternative molecular tagging methods need to be explored to enable more facile engineering of cells labeled with molecular specificity.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and s. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old, B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus. To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the upper-most meter of Mars.

Kminek, Gerhard↗
DOE OSTI2021

Diazotrophic Bacteria and Their Mechanisms to Interact and Benefit Cereals

Plant-growth-promoting bacteria (PGPB) stimulate plant growth through diverse mechanisms. In addition to biological nitrogen fixation, diazotrophic PGPB can improve nutrient uptake efficiency from the soil, produce and release phytohormones to the host, and confer resistance against pathogens. The genetic determinants that drive the success of biological nitrogen fixation in nonlegume plants are understudied. These determinants include recognition and signaling pathways, bacterial colonization, and genotype specificity between host and bacteria. This review presents recent discoveries of how nitrogen-fixing PGPB interact with cereals and promote plant growth. We suggest adopting an experimental model system, such as the Setaria–diazotrophic bacteria association, as a reliable way to better understand the associated mechanisms and, ultimately, increase the use of PGPB inoculants for sustainable agriculture. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biochemistry & Molecular Biology↗
DOE OSTI2020

Genome Sequences of Frankineae sp. Strain MT45 and Jatrophihabitans sp. Strain GAS493, Two Actinobacteria Isolated from Forest Soil

Frankiaceae are bacterial endosymbionts that are also found free-living in soil. Here, we present the genome sequences of two novel bacterial members of the order Frankiales , class Actinobacteria , isolated from temperate terrestrial forest soils. The genomes for MT45 and GAS493 indicate a genetic capacity for carbohydrate degradation but not nitrogen fixation.

DeAngelis, Kristen M.↗
NASA NTRS2012

Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously "shadowed" less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the "minority population" and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level. This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.

Vaishampayan, Parag A.↗
DOE OSTI2023

Genetic Fusion of Thermoresponsive Polypeptides with UCST–type Behavior Mediates 1D Assembly of Coiled–Coil Bundlemers

Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Furthermore, their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Scrambling Signal Modularity in Bottom-up Assembled Synthetic Pseudomonas Consortia Reveals Robust Information Transfer

There is immense potential in crafting synthetic microbial communities for application in human health, agriculture, the environment, and even biomanufacturing where an appropriately constructed consortium can be assembled with tremendous biosynthetic or degradative capabilities. In many of these cases, bacterial signaling serves as a form of intercellular information transfer that guides the collective’s behavior. Such communication is complex, as many signals, signal disruptors, microbial species, physical barriers, and spatiotemporal constraints may be involved. Here, in this work, we demonstrate that a multisignal pathway for molecular information transfer within a consortium of several Pseudomonas spp. can be scrambled (genetically and organizationally) while the original message is still effectively conveyed. Assembled from the bottom up, we have employed two types of signaling molecules (i) a redox active secondary metabolite (rhizospheric signal, phloroglucinol), and (ii) a bacterial quorum sensing signal (3-oxo-C12 acylhomoserine lactone, AI-1). These signals can be intraconverted and acted upon by designated community members. We show how the order in which the signals are received, transduced, and subsequently transmitted can be rearranged with minimal impact on the intended outcome. In the consortial context, we found this messaging structure can be remarkably robust. Inspired by rhizospheric molecular signaling mechanisms, this work provides a conceptual framework for designing signaling and information transfer processes within assembled communities.

Biological and medical sciences↗
DOE OSTI2022

The endosymbiotic box of protective tricks

This month’s Genome Watch underscores how genome and transcriptome sequencing of newly identified endosymbionts helps to connect genetic information to their protective functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Monomeric crystal structure of the vaccine carrier protein CRM 197 and implications for vaccine development

CRM 197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM 197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM 197 , where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM 197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM 197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Å resolution with the domain-swapping hinge loop (residues 379–387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer–dimer interconversion and for the optimization of conjugation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A plant genetic network for preventing dysbiosis in the phyllosphere

The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Here, the pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

BONCAT-Live for isolation and cultivation of active environmental bacteria

In diverse environments, microbes drive a myriad of processes, from geochemical and nutrient cycling to interspecies interactions, including associations with plants and animals. Their physiological state is dynamic and impacted by abiotic and biotic conditions, responding to environmental fluctuations by changes in cellular metabolism, according to their genetic potential. Molecular, cellular, and genomic approaches can identify and measure microbial responses and adaptation to environmental changes in native communities. However, isolating individual microbial cells that respond to specific changes for cultivation has been difficult. To address this, we implemented a novel bacterial isolation approach (BONCAT-Live) by integrating bio-orthogonal non-canonical amino acid tagging (BONCAT) in diverse native communities, with isolation and cultivation of cells responding to specific stimuli, at different time scales. In frozen Arctic permafrost samples, we identified and isolated dormant bacteria that become active after thawing under native or nutrient-enriched conditions. From the Populus tree rhizosphere, we isolated strains that thrive under high concentrations of root exudates that act as defense compounds and nutrients. In the human microbiome, we identified and isolated bacteria that rapidly proliferated when exposed to metabolites provided by the host or other co-occurring microbes. Further characterization of isolated bacterial strains will provide opportunities for in-depth determination of how these microbes adapt to changes in their environments, individually and as part of model communities.

Analytical Methods↗
DOE OSTI2020

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli

59 BASIC BIOLOGICAL SCIENCES↗