Biology subjects

Rivier, A.

Publications and source records attributed to Rivier, A..

5 recordsLinked to original sources

bioRxiv2026

Functional Genomics Reveals TNT Bioremediation Strategies in Pantoea sp. MT58 and Pseudomonas putida KT2440

2,4,6-Trinitrotoluene (TNT) is a recalcitrant and pervasive environmental pollutant. Although different environmental microbes have demonstrated their ability to degrade or transform TNT, the underlying genetic basis and cellular machinery remain unclear. In this study, we investigated bacterial strategies in response to TNT exposure in Pantoea sp. MT58 and P. putida KT2440 using proteomics and random barcode transposon-site sequencing (RB-TnSeq). Pantoea sp. MT58 was found to utilize TNT as a sole nitrogen source, whereas P. putida KT2440 exhibited only stress tolerance without assimilation. Pantoea sp. MT58 encodes multiple putative nitroreductases that were upregulated, yet deletion of these genes did not affect growth on TNT, revealing pathway redundancy. Furthermore, fitness profiling provided no evidence for genes involved in the canonical Meisenheimer-complex pathway associated with nitrite release. Instead, the data are most consistent with a sequential nitro-group reduction route in which nitrogen is ultimately recovered as ammonium, with nitrogen routed through the GS-GOGAT pathway with purine and urea pools as the candidate buffering architecture for TNT mineralization. Conversely, P. putida KT2440 relied on Ttg/RND efflux pumps and toluene tolerance proteins for survival without nitrogen assimilation from TNT. This work distinguishes routes for productive nitrogen assimilation from those involved in nitroaromatic tolerance, expanding the mechanistic understanding of anthropogenic compound metabolism to inform future bioremediation efforts.

microbiology↗

bioRxiv2025

Novel origin of replication for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Plasmids isolated or characterized from environmental samples serve as a resource that can be used to develop genetic tools for characterizing recently isolated or less-studied microbes. In this report, we leveraged sequences from a previously characterized groundwater plasmidome and developed a screen to identify novel plasmid origins. Putative origin sequences were used to construct a barcoded plasmid library, which contained both known and newly predicted origins. This library was tested against a panel of representative bacterial strains and led to the identification of 3 novel origins that putatively replicate in gram-negative bacteria not previously associated with these origin sequences. We empirically validated one of the newly identified origins, 6911, to be functional in both the model bacterial strain, Escherichia coli BW25113, as well as in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium from the widespread Pantoea genus. We confirmed that a plasmid bearing origin 6911 as the sole origin could replicate and had a copy number of 9 ({+/-} 2) in Pantoea sp. MT58. We successfully used a plasmid based on the new origin to express the reporter protein GFP, and two non-native metabolite pathways for the natural product, indigoidine and the terpenoid compound, isoprenol. By pairing functional novel origins of replication to non-model organisms this pipeline can expand the tool kit for genetic manipulations of both model and less-studied bacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/653210v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@45689corg.highwire.dtl.DTLVardef@d6daeeorg.highwire.dtl.DTLVardef@1bf4d06org.highwire.dtl.DTLVardef@59ccfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure.C_FLOATNO We developed a host-origin pair identification pipeline by constructing a plasmid library containing both literature-sourced and computationally predicted origins of replication. By conjugating this library into non-model microbes, we identified functional origins in diverse bacteria, and could leverage these findings to develop genetically tractable hosts for microbial engineering. C_FIG

microbiology↗

bioRxiv2025

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida, a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putidas native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a [~]16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing [~]900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643695v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@533605org.highwire.dtl.DTLVardef@ff98fforg.highwire.dtl.DTLVardef@9592fdorg.highwire.dtl.DTLVardef@1e52993_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs associate closely with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrate genome editing in Klebsiella michiganensis M5al. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/595406v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@ea73a4org.highwire.dtl.DTLVardef@4e540eorg.highwire.dtl.DTLVardef@706557org.highwire.dtl.DTLVardef@10d4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Conservation of nitrogenase functionality over long timescales

The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, that is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism. IMPACT STATEMENTThe enzymatic mechanism for dinitrogen reduction is an ancient feature of nitrogenases that persisted over hundreds of millions of years.

evolutionary biology↗