Biology subjects

Michener, J. K.

Publications and source records attributed to Michener, J. K..

13 recordsLinked to original sources

bioRxiv2026

Horizontal transfer of chromosomal DNA mediated by an integrative and conjugative element generates frequent localized recombination in Novosphingobium aromaticivorans

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that recombination between soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional, more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed into strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans, increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. ImportanceHorizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that ICE-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.

microbiology↗

bioRxiv2026

Structural and Oligomeric Characterization of Substrate- and Product-selective Nylon Hydrolases

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

biochemistry↗

bioRxiv2026

Parallel tracking by sequencing reveals the impact of restriction modification systems on transfer of an integrative and conjugative element

Horizontal gene transfer (HGT) is a promising avenue for microbiome engineering that enables DNA delivery to microbes in their native environment. Integrative and conjugative elements (ICEs), a class of genomically-integrated mobile elements, are ideal vectors for this purpose. Developing targeted ICE transfer for controlled microbiome engineering requires a better understanding of ICE mobility in complex communities. However, current methods for tracking horizontal gene transfer are not high throughput. To improve upon current methods, we developed a sequencing-based strategy to track and quantify ICE transfer in complex mixtures that we refer to as ICE-seq. This method was able to assess ICE transfer in a synthetic community of 150 recipients simultaneously and in combination with phenotypic assays demonstrated that restriction-modification (RM) systems in the donor and recipient alter ICE transfer rate with subspecies resolution by up to 10,000-fold. We propose that manipulating RM systems can be a strategy to engineer targeted ICE transfer for directed microbiome engineering. ImportanceTypical approaches to engineer microbiomes involve adding beneficial strains to existing microbiomes, for example the addition of probiotics to the human gut microbiome or soil amendments to improve plant health or for bioremediation. However, these beneficial bacteria often do not survive for extended periods of time, which limits their overall effectiveness. To circumvent this issue, we can transfer beneficial genes by native mechanisms into existing bacteria that are already in the target environment where they can then perform the beneficial function encoded by these genes. In this work, we developed a technique to track gene transfer into multiple recipients simultaneously. Furthermore, bacteria have innate defense systems to protect against invading DNA that can reduce this gene transfer. Our technique allowed us to identify the impact of these defense systems and their potential function for targeting gene transfer to specific recipients.

microbiology↗

bioRxiv2025

Process optimization for enhanced enzymatic nylon deconstruction

Plastics such as polyamides (PAs) possess unique physicochemical properties that make them indispensable in modern society. However, their energy-intensive production and challenging end-of-life management highlight the urgent need for efficient recycling or remanufacturing solutions. Enzymatic depolymerization offers a promising route toward circular recycling, yet remains constrained by limited enzyme characterization, lack of validation under industrially relevant conditions, and overall performance. Here we optimized the reaction conditions for three recently discovered nylon-degrading enzymes. One of them, Nyl12, achieved product titers with PA6 and PA66 that exceed previously reported values, without enzyme engineering or substrate pretreatment. We further demonstrated the scalability of the process and its application to complex PA-based materials used in microelectronic components. Analysis of substrate features, including surface area and particle size, revealed key parameters governing enzymatic activity and provided a framework for future pretreatment and process optimization efforts. In combination, these efforts provide a new benchmark for enzymatic nylon recycling.

biochemistry↗

bioRxiv2025

Regioselective biosynthesis of oligoamides as precursors for sequence-controlled co-polyamides

Polyamides are important natural and synthetic polymers, best exemplified by proteins and nylons respectively. Proteins demonstrate that novel polymers with emergent properties can be generated by combining diverse monomers in precisely defined sequences. However, commercial polyamides represent only a small fraction of the potential diversity in polyamide sequences, due to the synthetic challenges of sequence-controlled polymerization. Amide synthetases have been shown to synthesize a broad array of nylon-relevant diads, but the generation of novel sequenced copolyamides requires enzymes capable of acting with longer and more diverse substrates. In this study, we demonstrated that NRPS-independent siderophore (NIS) synthetases, represented by DesD, can ligate oligomeric substrates. A simultaneous enzyme cascade using DesD enabled the synthesis of an oligotriad directly from unprotected substrates. Moreover, the regioselectivity of DesD allowed the selective synthesis of a sequenced amide tetrad, the precursor to a novel sequenced-defined polyamide with properties superior to nylon 66. This study establishes a direct biocatalytic route for the facile synthesis of sequenced oligoamides from unprotected bifunctional substrates, opening new possibilities to synthesize sequenced copolyamides.

biochemistry↗

bioRxiv2025

Genome shuffling enables quantitative trait locus mapping in Bacillus subtilis

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size ([~]10 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria. One-Sentence SummaryBacterial genome shuffling mimics sexual recombination and enables genetic mapping of complex phenotypes to causal alleles.

microbiology↗

bioRxiv2025

Enzymatic synthesis of diverse oligoamide polymer precursors

Nylons, a major class of synthetic polyamides, are widely used due to their excellent mechanical strength, thermal stability, and chemical resistance. Conventional nylon production relies on the polymerization of lactams or stoichiometric nylon salts. However, applying these approaches to unconventional precursors such as bio-derived glutaric acid produces polymers with low molecular weight and limited applications. To address these challenges, we demonstrated that chemically-synthesized nylon diads enable the production of higher-molecular-weight polyamides compared with traditional salts. We then identified a biosynthetic approach using amide synthetases to convert unprotected bifunctional substrates into nylon-relevant diads. Using a cofactor regeneration system, enzymatic diad synthesis was scaled to produce sufficient material for laboratory-scale characterization and solid-state polymerization. Amide synthetases demonstrated broad substrate scope, catalyzing the regioselective assembly of diverse nylon-relevant diacids, diamines, and {omega}-amino acids. This strategy offers a novel route to synthesize challenging nylon monomers and advances production of bioderived nylons.

biochemistry↗

bioRxiv2024

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases. SignificanceNylons are common industrial polyamides with few recycling options. As an alternative to mechanical or chemical recycling, enzymes may provide a selective and energy-efficient route to deconstruct nylons from mixed waste. Several nylon hydrolases have been identified, most notably NylC, but these enzymes are all closely related and demonstrated similar activity and substrate range. In this work, we investigated a diverse set of enzymes and showed that nylon hydrolase activity is common, providing new insights into the evolution of microbial nylon hydrolysis. Unlike NylC, several enzymes demonstrated unprecedented substrate selectivity, preferentially hydrolyzing Nylon 6,6 compared to Nylon 6. These enzymes can be used to understand substrate selectivity in nylon hydrolysis and to engineer enzymes for nylon recycling from mixed waste.

biochemistry↗

bioRxiv2024

Laboratory evolution in Novosphingobium aromaticivorans enables rapid catabolism of a model lignin-derived aromatic dimer

Lignin contains a variety of interunit linkages, which leads to a range of potential decomposition products that can be used as carbon sources by microbes. {beta}-O-4 linkages are the most common in native lignin and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from {beta}-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a {beta}-O-4 linkage, guaiacylglycerol-{beta}-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide mutation in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, {beta}-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds. ImportanceLignin degradation is a key step for both carbon cycling in nature and biomass conversion to fuels and chemicals. Bacteria can catabolize lignin-derived aromatic compounds, but the complexity of lignin means that full mineralization requires numerous catabolic pathways and often results in slow growth. Using experimental evolution, we identified a new enzyme for catabolism of a lignin-derived aromatic monomer, {beta}-hydroxypropiovanillone. A single mutation in the promoter of the associated gene significantly increased bacterial growth with either {beta}-hydroxypropiovanillone or a related lignin-derived aromatic dimer. This work expands the repertoire of known aromatic catabolic genes and demonstrates that slow catabolism of lignin-derived aromatic compounds may be due to misregulation under laboratory conditions rather than inherent catabolic challenges.

microbiology↗

bioRxiv2024

Catabolic pathway acquisition by soil pseudomonads readily enables growth with salicyl alcohol but does not affect colonization of Populus roots

Horizontal gene transfer (HGT) is a fundamental evolutionary process that plays a key role in bacterial evolution. The likelihood of a successful transfer event is expected to depend on the precise balance of costs and benefits resulting from pathway acquisition. Most experimental analyses of HGT have focused on phenotypes that have large fitness benefits under appropriate selective conditions, such as antibiotic resistance. However, many examples of HGT involve phenotypes that are predicted to provide smaller benefits, such as the ability to catabolize additional carbon sources. We have experimentally reproduced one such HGT event in the laboratory, studying the effects of transferring a pathway for catabolism of the plant-derived aromatic compound salicyl alcohol into soil isolates from the Pseudomonas genus. We find that pathway acquisition enables rapid catabolism of salicyl alcohol with only minor disruptions to existing metabolic and regulatory networks of the new host. However, this new catabolic potential does not confer a measurable fitness advantage during competitive growth in the rhizosphere. We conclude that the phenotype of salicyl alcohol catabolism is readily transferred by HGT but is selectively neutral under environmentally-relevant conditions. We propose that this condition is common and that HGT of many pathways will be self-limiting, because the selective benefits are small and negative frequency-dependent.

microbiology↗

bioRxiv2020

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Computational analyses suggested that this bias is due to the action of restriction-modification systems whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that the intensity of recombination is uniform across the genome without concentration into hotspots. Unexpectedly, our results revealed that large species-level genetic distance did not affect key recombination parameters. Our study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

microbiology↗

bioRxiv2018

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

Metabolic pathways are frequently transferred between bacterial strains in the environment through horizontal gene transfer (HGT), yet laboratory engineering to introduce new metabolic pathways often fails. Successful use of a pathway requires co-evolution of both pathway and host, and these interactions may be disrupted upon transfer to a new host. Here we show that two different pathways for catabolism of coumarate failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly-acquired pathways and the modifications required to overcome these limitations. Both pathways required mutations to the host to enable effective growth with coumarate, but the necessary mutations differed depending on the chemistry and intermediates of the pathways. In one case, an intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid mutations to IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is already resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. These discoveries will aid in our understanding of HGT and ability to predictably engineer metabolism.\n\nThis manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

microbiology↗

bioRxiv2018

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. These challenges are amplified when the enzymes are expressed at single copy from the chromosome. We have explored these issues using 4-hydroxybenzoate monooxygenase homologs heterologously expressed in Escherichia coli. Initial chromosomal enzyme expression was insufficient to support consistent growth with 4-hydroxybenzoate. Experimental evolution identified mutations that improved pathway activity. One set of mutations was common between homologs, while a second class of mutations was homolog-specific. Ultimately, we were able to identify a set of mutations that provided sufficient activity for growth with 4-hydroxybenzoate while maintaining or improving growth with protocatechuate. These findings demonstrate the potential for flexible, scalable chromosomal pathway engineering, as well as the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

bioengineering↗