Biology subjects

Donohue, T. J.

Publications and source records attributed to Donohue, T. J..

10 recordsLinked to original sources

bioRxiv2025

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Hamptons Road Sanitation District (HRSD) with High and Low Dissolved Oxygen Conditions

Aeration in biological nutrient removal (BNR) systems constitutes one of the largest energy demands in water resource recovery facilities (WRRFs). Previous studies have shown that lowering dissolved oxygen (DO) concentrations can sustain effective nitrification and phosphorus removal while substantially reducing energy consumption. However, the microbial mechanisms enabling these low-DO processes remain poorly understood. In this study, we investigated microbial communities associated with reduced DO in a pilot-scale BNR system operated by the Hampton Roads Sanitation District (HRSD). DO was reduced over an 18-month period from 2.5 mg/L to 0.2 mg/L. Metagenomic DNA was obtained from samples from each DO condition then sequenced using PacBio HiFi technology. A total of 316 metagenome-assembled genomes were recovered and after dereplication, 207 were found to be unique. These data augment the metagenomic information related to wastewater treatment under low-DO conditions and provide valuable resources for understanding microbial adaptation to oxygen-limited BNR operation.

genomics↗

bioRxiv2025

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Los Angeles County Sanitation District (LACSD) with High and Low Dissolved Oxygen Conditions

Aeration represents one of the largest energy costs in water resource recovery facilities (WRRFs). Previous studies have shown that effective nitrification and phosphorus removal can be maintained in biological nutrient removal (BNR) systems operated under low dissolved oxygen (DO) conditions, significantly reducing energy use. To improve understanding of microbial community adaptation to reduced DO, we analyzed metagenomes and metagenome-assembled genomes (MAGs) from the Pomona WRRF (Los Angeles County Sanitation Districts) before and after a gradual decrease in operating DO from approximately 3.5 mg/L to 0.7 mg/L over 18 months. Metagenomic DNA was isolated from high and low DO samples, then sequenced using PacBio HiFi technology. A total of 492 MAGs were recovered of which 304 were unique after dereplication. These genomes expand current knowledge of microbial community dynamics in low-DO BNR systems and provide valuable genomic resources for understanding microbial adaptation to energy-efficient wastewater treatment processes.

genetics↗

bioRxiv2023

Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria. IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria, Novosphingobium aromaticivorans and Rhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.

microbiology↗

bioRxiv2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

genetics↗

bioRxiv2020

Functional pathway redundancy in the metabolism of plant-derived phenolics by Novosphingobium aromaticivorans

Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds by N. aromaticivorans. The results showed the involvement of DesA in O-demethylation of syringic and vanillic acids, LigM in O-demethylation of vanillic acid and 3-MGA, and a new O-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O-demethylation of 3-MGA to GA. We predict this pathway channels [~]15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCEFor lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

microbiology↗

bioRxiv2020

Rewiring the specificity of extra-cytoplasmic function sigma factors

SUMMARYBacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators’ DNA binding and precise determination of biologically significant binding sites. Here, we address these challenges by solving the DNA-specificity code of extra-cytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the auto-regulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid – nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic foot-printing pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that ECF σs play varied roles: some are global regulators controlling many genes throughout the genome that are important under many conditions, while others are local regulators, controlling few closely linked genes in response to specific stimuli. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2019

Metabolic models of mixed culture fermentation diagnose bottlenecks and predict strategies for improved medium-chain fatty acid production

Multi-species microbial communities determine the fate of materials in the environment and can be harnessed to produce beneficial products from renewable resources. In a recent example, fermentations by microbial communities have produced medium-chain fatty acids (MCFAs). Tools to predict, assess, and improve the performance of these communities, however, are limited. To provide such tools, we constructed two metabolic models of MCFA-producing microbial communities based on available genomic, transcriptomic and metabolomic data. The first model is a unicellular model (iFermCell215), while the second model (iFermGuilds789) separates fermentation activities into functional guilds. Ethanol and lactate are fermentation products known to serve as substrates for MCFA production, while acetate is another common co-metabolite during MCFA production. Simulations with iFermCell215 predict that low molar ratios of acetate-to-ethanol favor MCFA production, whereas the products of lactate and acetate co-utilization are less dependent on the acetate-to-lactate ratio. In simulations of an MCFA-producing community fed a complex organic mixture derived from lignocellulose, iFermGuilds789 predicted that lactate was an extracellular co-metabolite that served as a substrate for butyrate (C4) production. Extracellular hexanoic (C6) and octanoic acids (C8) were predicted by iFermGuilds789 to be from community members that directly metabolize sugars. Modeling results provide several hypotheses that can improve our understanding of microbial roles in a MCFA-producing microbiome and inform strategies to increase MCFA production. Further, these models represent novel tools for exploring the role of mixed microbial communities in carbon recycling in the environment, as well as on beneficial reuse of organic residues. IMPORTANCEMicrobiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

microbiology↗

bioRxiv2019

Anaerobic degradation of syringic acid by an adapted strain of Rhodopseudomonas palustris

While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogenous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a byproduct of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions, via the benzoyl-CoA degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619-rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris.\n\nIMPORTANCELignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for production of aromatic-based biochemicals requires detailed knowledge of metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveals a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire to metabolize aromatic compounds from lignin and other renewable sources.

microbiology↗

bioRxiv2019

Multi-omic analysis of medium-chain fatty acid synthesis by Candidatus Weimerbacter bifidus, gen. nov., sp. nov., and Candidatus Pseudoramibacter fermentans, sp. nov.

Chain elongation is emerging as a bioprocess to produce valuable medium-chain fatty acids (MCFA; 6 to 8 carbons in length) from organic waste streams by harnessing the metabolism of anaerobic microbiomes. Although our understanding of chain elongation physiology is still evolving, the reverse {beta}-oxidation pathway has been identified as a key metabolic function to elongate the intermediate products of fermentation to MCFA. Here, we describe two chain-elongating microorganisms that were enriched in an anaerobic microbiome transforming the residues from a lignocellulosic biorefining process to short- and medium-chain fatty acids. Based on a multi-omic analysis of this microbiome, we predict that Candidatus Weimerbacter bifidus, gen. nov., sp. nov. used xylose to produce MCFA, whereas Candidatus Pseudoramibacter fermentans, sp. nov., used glycerol and lactate as substrates for chain elongation. Both organisms are predicted to use an energy conserving hydrogenase to improve the overall bioenergetics of MCFA production.\n\nIMPORTANCEMicrobiomes are vital to human health, agriculture, environmental processes, and are receiving attention as biological catalysts for production of renewable industrial chemicals. Chain elongation by MCFA-producing microbiomes offer an opportunity to produce valuable chemicals from organic streams that otherwise would be considered waste. However, the physiology and energetics of chain elongation is only beginning to be studied, and we are analyzing MCFA production by self-assembled communities to complement the knowledge that has been acquired from pure culture studies. Through a multi-omic analysis of an MCFA-producing microbiome, we characterized metabolic functions of two chain elongating bacteria and predict previously unreported features of this process.

microbiology↗

bioRxiv2018

Metatranscriptomic and thermodynamic insights into medium-chain fatty acid production using an anaerobic microbiome

Biomanufacturing from renewable feedstocks represents a sustainable strategy to offset fossil fuel-based chemical production. One potential biomanufacturing strategy is production of medium chain fatty acids (MCFA) from organic feedstocks using either pure cultures or microbiomes. While the set of microbes in a microbiome can often metabolize more diverse organic materials than a single species, and the role of specific species may be known, knowledge of the carbon and energy flow within and between organisms in MCFA producing microbiomes is only starting to emerge. Here, we integrate metagenomic, metatranscriptomic, and thermodynamic analyses to predict and characterize the metabolic network of an anaerobic microbiome producing MCFA from organic matter derived from lignocellulosic ethanol fermentation conversion residue. A total of 37 high quality (>80% complete, < 10% contamination) metagenome-assembled genomes (MAGs) were recovered from the microbiome and metabolic reconstruction of the 10 most abundant MAGs was performed. Metabolic reconstruction combined with metatranscriptomic analysis predicted that organisms affiliated with Lactobacillus and Coriobacteriaceae degraded carbohydrates and fermented sugars to lactate and acetate. Lachnospiraceae and Eubacteriaceae affiliated organisms were predicted to transform these fermentation products to MCFA. Thermodynamic analyses identified conditions in which H2 is expected to be either produced or consumed, suggesting a potential role of H2 partial pressure on MCFA production. From an integrated systems analysis perspective, we propose that MCFA production could be improved if microbiomes are engineered to use homofermentative instead of heterofermentative Lactobacillus, and if MCFA-producing organisms are engineered to preferentially use a thioesterase instead of a CoA transferase as the terminal enzyme in reverse {beta}-oxidation.\n\nImportanceMixed communities of microbes play important roles in health, the environment, agriculture, and biotechnology. While tapping the combined activity of organisms within microbiomes may allow the utilization of a wider range of substrate over the use of pure cultures for biomanufacturing, harnessing the metabolism of these mixed cultures remains a major challenge. Here, we predict metabolic functions of bacteria in a microbiome that produces medium-chain fatty acids from a renewable feedstock. Our findings lay the foundation to begin addressing how to engineer and control microbiomes for improved biomanufacturing; to build synthetic mixtures of microbes that produce valuable chemicals from renewable resources; and to better understand microbial communities that contribute to health, agriculture, and the environment.

systems biology↗