Biology subjects

Van Fossen, E. M.

Publications and source records attributed to Van Fossen, E. M..

4 recordsLinked to original sources

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

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii

Sorghum (Sorghum bicolor) is a major food and bioenergy grass species cultivated worldwide. To promote more robust and sustainable growth of this important crop, we need a deeper understanding of the plant-microbe interactions between sorghum and soil microbial communities that benefit plant host resiliency and enhance nutrient acquisition. The release of specific metabolites from plant roots, or root exudation, drives these plant-microbe interactions, but the molecular pathways by which root exudates shape the sorghum rhizosphere microbiome require further elucidation. To investigate these complex interkingdom interactions in the sorghum rhizosphere, we developed a photoaffinity probe based on sorgoleone, a hydrophobic secondary metabolite and allelochemical produced in sorghum seedling root exudates. Here, we apply a new synthetic sorgoleone diazirine alkyne photoaffinity probe (SoDA-PAL) to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth promoting microbe derived from Sorghum bicolor rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins that were complementary to a previously identified gene cluster involved in sorgoleone catabolism. Proteins identified by SoDA-PAL included a select set of putative transporters, transcription regulators, and a subset of proteins with lipid and secondary metabolic activities. We confirm binding of SoDA-PAL to a putative hydrolase in the /{beta} fold family (OH685_09420) through structural bioinformatics and in-vitro recombinant protein analysis. This photoaffinity labeling approach using metabolite-based probes can be extended in the future to proteomic profiling of complex rhizosphere microbiomes to discover genes that can be leveraged to promote beneficial plant-microbe interactions. ImportanceHere we demonstrate a photoaffinity-based chemical probe modeled after sorgoleone, a known secondary metabolite released from the roots of sorghum, can be used to dissect complicated plant-microbe interactions. Applying this probe to the sorghum-associated bacterium Acinetobacter pittii identified diverse proteins that directly interact with sorgoleone. We show that probe labeling is dose-dependent and is sensitive to competition with purified sorgoleone, demonstrating the probe is selective for protein targets that directly interact with sorgoleone. By using the probe to broadly profile proteins that interact with sorgoleone, we identified bacterial catabolic pathways, unintuitive transcriptional regulation pathways, and vital exchange mechanisms involving transporters that may be involved in sorgoleone utilization and cellular response toward this plant metabolite. We envision that this workflow will expand our understanding of the sorghum root exudate interactome and elucidate the molecular mechanisms by which specific metabolites shape the sorghum rhizosphere microbiome.

biochemistry↗