Biology subjects

Egbert, R.

Publications and source records attributed to Egbert, R..

11 recordsLinked to original sources

bioRxiv2026

Rational reduction of a sorghum SynCom that preserves growth promotion reveals flavonoid-mediated plant microbe interactions

O_LIPlant growth is influenced by the composition of its associated microbiome. The inherent complexity and functional redundancy of natural plant microbiomes presents a formidable barrier to understanding the myriad biological interactions therein. Efforts have been made to develop synthetic microbial communities (SynComs) that can provide a rigorous and generalizable framework for the rational design of next-generation microbial products for sustainable agriculture. We test multiple strategies for stable, plant growth promoting SynCom design and evaluate the phenotypic and molecular impacts of a successful plant-SynCom interaction. C_LIO_LIWe designed 4 distinct, reduced-complexity variants of SynCom SRC1 and assessed their capacities for colonization, stability, and plant growth promotion. To understand the impact on plant performance of our highest performing SynCom variant, we characterized the hosts longitudinal transcriptional response to SynCom inoculation and corroborated the results with metabolomics analysis. C_LIO_LIThe top performing SynCom stably colonized sorghum roots and rhizospheres, elicited plant growth promotion, and induced dynamic spatiotemporal gene transcription in sorghum roots and shoots defined by modulation of growth-defense tradeoff machinery and enhanced flavonoid production. C_LIO_LIThe resultant reduced-complexity SynCom is a highly stable, soil-independent, plant growth promoting, and demonstrates the utility of colonization-based selection criteria, integrated with longitudinal transcriptomic and metabolomic characterization. C_LI

plant biology↗

bioRxiv2025

RhizoGrid: Illuminating Spatial Rhizosphere Dynamics

The rhizosphere microbiome directly influences plant health and acclimation to extreme environments, yet plant-microbe interactions in the rhizosphere have proven complex and difficult to study. We present RhizoGrid, a new methodological framework that integrates a 3D-printed pot structure with spatial measurements of root structure, metabollite and taxonomy to detect links between metabolites and microbes along a soil-grown root system. The RhizoGrid identifies microhabitats hidden belowground. Using the food, forage, and bioenergy crop sorghum, we showcase how the RhizoGrid opens new frontiers for exploring the heterogeneity and complexity of interactions between root exudates and microbes in the rhizosphere.

systems biology↗

bioRxiv2025

Single-Molecule Imaging Reveals Transcription-Driven Supercoiling in Unconstrained DNA

In vitro studies of supercoiling dynamics have relied on externally applied force to twist constrained DNA. It is thus unknown whether transcription alone can generate supercoiling in topologically unconstrained DNA, and whether RNAP complexes could act as the topological barriers required to confine this stress. Here, using single-molecule imaging of 20k base pair long double strand DNA, we reveal that RNAP dynamically generates and confines supercoiling in unconstrained DNA. We demonstrate that multiple transcription events create transient topological domains, which allow plectonemes to stabilize between them; in contrast, a single transcription event does not lead to plectoneme formation. Furthermore, we show that this transcription-induced supercoiling is modulated by topoisomerase activity. Crucially, by observing that RNAP itself provides sufficient topological barriers, we establish that transcription-induced supercoiling is an inherently localized phenomenon. These findings redefine the physical basis of transcription-coupled supercoiling, providing a new mechanistic framework for modeling gene regulation based on local topology, with broader implications for genome editing and genome evolution.

molecular biology↗

bioRxiv2025

Reinforced CRISPR interference enables reliable multiplex gene repression in phylogenetically distant bacteria

Genetic screens are essential for uncovering novel molecular mechanisms and identifying the functions of hypothetical proteins. CRISPR interference (CRISPRi) is a powerful, programmable, and sequence-specific gene repression technology that can be used for high-throughput screening and targeted gene repression. Despite its ease of use, the initial development of CRISPRi systems is labor-intensive in many non-model organisms. Our goal is to simplify this by establishing a host-agnostic CRISPRi platform that utilizes the serine recombinase-assisted genome engineering (SAGE) system. This system integrates CRISPRi machinery directly into the bacterial chromosome, overcoming the limitations of plasmid-based systems and enabling wide sharing across diverse bacteria. We demonstrate the design and optimization of multiplexed CRISPRi to repress multiple genes simultaneously in phylogenetically distant bacteria. We use a Francisella novicida-derived Cas12a system that processes multiple distinct CRISPR RNAs, each targeting a unique gene sequence, from a single transcript. This allows easy multi-gene repression. By reinforcing gene repression with multiple guides targeting a single gene, we achieve robust genetic perturbations without the need to pre-screen the efficacy of guide RNAs. Using this toolkit, we perturb multiple combinations of growth and visual phenotypes in Pseudomonas fluorescens and demonstrate simultaneous repression of multiple fluorescent proteins to near background levels in bacteria from various other genera. While the tools are directly portable to all SAGE-compatible microbes, we illustrate the utility of SAGE by optimizing CRISPRi performance in Rhodococcus jostii through a combinatorial screen of Cas protein and CRISPR array expression variants. The efficient integration of CRISPRi machinery via the SAGE system paves the way for versatile genetic screening, enabling profound insights into gene functions both in laboratory conditions and relevant naturalistic scenarios.

microbiology↗

bioRxiv2025

Efficient genetic code expansion tools enable in vivo study of lysine acetylation in non-model bacteria

Recent proteomic advancements have revealed widespread N{varepsilon}-lysine acetylation in pathways governing pathogenicity, metabolism, and antibiotic resistance in bacteria. The spontaneous, non-specific nature of this modification in prokaryotes obscures its biological role, necessitating prokaryotic specific in vivo interrogation systems. Genetic Code Expansion (GCE) offers a powerful method to investigate the roles and regulation dynamics of acetyl-lysine in vivo with the precise incorporation of a suite of non-canonical amino acids, including acetyl-lysine analogs. However, its use has been largely restricted to E. coli strains due to challenges associated with implementation and optimization of the technology in more diverse bacterial strains. Here, we present a bacterial host-agnostic, readily optimizable GCE platform designed to site-specifically incorporate non-canonical amino acids into target proteins within living bacteria. We further demonstrate the versatility of this technology by showcasing, for the first time, the successful incorporation of acetyl-lysine in a non-E. coli bacterium.

synthetic biology↗

bioRxiv2023

A novel phenylpropanoid methyl esterase enables catabolism of aromatic compounds that inhibit biological nitrification

Agriculture is the largest source of greenhouse gases (GHG) production. Conversion of nitrogen fertilizers into more reduced forms by microbes through a process known as biological nitrification drives GHG production, enhances proliferation of toxic algal blooms, and increases cost of crop production. Some plants reduce biological nitrification in soils by exuding a diverse array of biological nitrification inhibitors (BNIs) that inhibit the ammonium oxidizing microbes responsible for nitrification. Applying synthetic biology to enhance and transfer BNI production into food and bioenergy crops is a promising approach to reduce nitrification but the success of this strategy is contingent upon improving our limited understanding of BNIs mechanisms-of-action and degradation in the soil. We addressed this gap by investigating the previously unknown metabolic route through which a prominent class of aromatic BNIs known as phenylpropanoid methyl esters (PPMEs) are catabolized. While neither transcriptomics (RNAseq) or high-throughput functional genomics (RB-TnSeq) methods reduced the genetic search space for pathway discovery into a tenable number of genes, the combination of narrowed the search space to a collection of 4 proteins of unknown function. Using genetic and biochemical analyses we found that two previously uncharacterized enzymes, including a novel phenylpropanoid methyl esterase, funnel PPMEs into an established phenylpropanoid catabolism pathway. Transfer of these two enzymes into bacteria capable of using other phenylpropanoids use of PPMEs as carbon sources. This work both provided insight into BNI catabolism and is the first step towards development of model in vivo plant-microbe systems for studying BNI mechanisms under well controlled conditions.

microbiology↗

bioRxiv2023

Defined synthetic microbial communities colonize and benefit field-grown sorghum

The rhizosphere represents a dynamic and complex interface between plant hosts and the microbial community found in the surrounding soil. While it is recognized that manipulating the rhizosphere has the potential to improve plant fitness and health, engineering the rhizosphere microbiome through inoculation has often proved challenging. This is in large part due to the competitive microbial ecosystem in which the added microbes must survive, and lack of adaptation of these added microbes to the specific metabolic and environmental pressures of the rhizosphere. Here, we constructed an inoculation formula using a defined synthetic community (dSynCom) approach that we hypothesized would improve engraftment efficiency and potentially the relationship with the host plant, Sorghum bicolor. The dSynCom was assembled from bacterial isolates that were either: 1) identified to potentially play a role in community cohesion through network analysis, or 2) identified to benefit from host-specific exudate compounds. Growth of the dSynCom was first evaluated in vitro on solid media, secondly in planta under gnotobiotic laboratory conditions, and finally using sorghum plants grown in the field. We demonstrate that the dSynCom performs best in terms of maintaining diversity when grown in the presence of the plant host in lab conditions, and that many lineages are lost from the community when grown either in vitro or in a native field setting. Finally, we demonstrate that the dSynCom is able to promote growth of above- and below-ground plant phenotypes compared to uninoculated controls, both in the lab and when applied to plants grown in the field. These results demonstrate the potential utility of SynComs for supporting crop performance even in the absence of persistence, and the need for a deeper mechanistic understanding of community control of host fitness in agricultural contexts.

synthetic biology↗

bioRxiv2023

Sorgoleone degradation by sorghum-associated bacteria; an opportunity for enforcing plant growth promotion

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe. SignificanceThe grain crop sorghum exudes an herbicidal compound called sorgoleone from its root tips, which inhibits the growth of other plants. We isolated bacteria that grow on sorogleone and identified a cluster of bacterial genes required for sorogleone degradation that can be used as a biomarker for this trait. An approach to improve the production of crops in stressful conditions such as drought, is to encourage their association with plant growth promoting bacteria. Our discovery of sorgoleone degradation genes opens the door to engineering bacteria that receive benefit from sorghum in the form of a plant-specific growth substrate, and in return promote the growth of this crop.

microbiology↗

bioRxiv2020

Resource reallocation in engineered Escherichia coli strains with reduced genomes

A major challenge in synthetic biology is properly balancing evolved and engineered functions without compromising microbial fitness. Many microbial proteins are not required for growth in regular laboratory conditions, but it is unclear what fraction of the proteome can be eliminated to increase bioproduction and maintain fitness. Here, we investigated the effects of massive genome reduction in E. coli on the expression level and evolutionary stability of a model biosynthetic pathway to produce the pigment protodeoxyviolacein (PDV). We identified an amino acid metabolism imbalance and compromised growth that were correlated with elimination of genes associated with significant proteome fraction. Proteomic profiling suggested that increased amino acid pools are responsible for an alleviation of fitness defects associated with PDV expression. In addition, all strains with genome reductions that significantly affected the proteome exhibited decreased stability of PDV production compared to the wild-type strain under persistent PDV expression conditions despite the alleviation of fitness defects. These findings exhibit the importance of balancing evolved functions with engineered ones to achieve an optimal balance of fitness and bioproduction.

bioengineering↗

bioRxiv2020

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural purposes. Synthetic biology can address these limitations, but its application is limited by availability of appropriate tools for sophisticated, high-throughput genome engineering that function in environments where selection for DNA maintenance is impractical. Here we present an orthogonal, Serine-integrase Assisted Genome Engineering (SAGE) system, which enables iterative, site-specific integration of up to 10 different DNA constructs at efficiency on par or better than replicating plasmids. SAGE does not require use of replicating plasmids to deliver recombination machinery, and employs a secondary serine-integrase to excise and recycle selection markers. Furthermore, unlike the widely utilized pBBR1 origin, DNA transformed using SAGE is stable without selection. We highlight SAGEs utility by constructing a 287-member constitutive promoter library with a [~]40,000-fold dynamic range in P. fluorescens SBW25. We show that SAGE functions robustly in diverse - and {gamma}-proteobacteria, thus providing evidence that it will be broadly useful for engineering industrial or environmental bacteria.

synthetic biology↗

bioRxiv2016

Self-destructive altruism in a synthetic developmental program enables complex feedstock utilization

Stochastic differentiation and programmed cell death are common developmental processes in microbes, driving diverse altruistic behaviors that promote cooperation. Utilizing cell death in developmental programs requires control over the rate of differentiation to balance cell proliferation against the utility of sacrifice. However, the regulatory networks that control these behaviors are often complex and have yet to be successfully harnessed as biotechnology. Here, we engineered a synthetic developmental gene network that couples stochastic differentiation with programmed cell death to implement a two-member division of labor. Progenitor cellobiose consumer cells were engineered to grow on cellobiose and differentiate at a controlled rate into self-destructive altruists that release an otherwise sequestered cellulase enzyme payload through autolysis to form a developmental Escherichia coli consortium that utilizes cellulose for growth. We used an experimentally parameterized model of task switching, payload delivery and cellulose conversion to nutrients to set key parameters to achieve overall population growth supported by cellulase release, liberating 14-23% of the available carbon. An inevitable consequence of engineering altruistic developmental behaviors is the emergence of cheaters that undermine cooperation. We observed cheater phenotypes for consumers and altruists, identified mutational hotspots and constructed a predictive model of circuit longeivity based on mutation rate estimates for each mode of evolutionary escape. This work introduces the altruistic developmental program as a new tool for synthetic biology, demonstrates the utility of population dynamics models to engineer complex phenotypes and provides a testbed for probing the evolutionary biology of self-destructive altruism.

synthetic biology↗