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DOE OSTI2019

Bacterial Tradeoffs in Growth Rate and Extracellular Enzymes

Like larger organisms, bacteria possess traits, or phenotypic characteristics, that influence growth and impact ecosystem processes. Still, it remains unclear how these traits are organized across bacterial lineages. Using 49 bacterial strains isolated from leaf litter in Southern California, we tested the hypothesis that bacterial growth rates trade off against extracellular enzyme investment. We also tested for phylogenetic conservation of these traits under high and low resource conditions represented, respectively, by Luria broth (LB) and a monomer-dominated medium extracted from plant litter. In support of our hypotheses, we found a negative correlation between the maximum growth rate and the total activity of carbon-, nitrogen-, and phosphorus-degrading extracellular enzymes. However, this tradeoff was only observed under high resource conditions. We also found significant phylogenetic signal in maximum growth rate and extracellular enzyme investment under high and low resource conditions. Driven by our bacterial trait data, we proposed three potential life history strategies. Resource acquisition strategists invest heavily in extracellular enzyme production. Growth strategists invest in high growth rates. Bacteria in a third category showed lower potential for enzyme production and growth, so we tentatively classified them as maintenance strategists that may perform better under conditions we did not measure. These strategies were related to bacterial phylogeny, with most growth strategists belonging to the phylum Proteobacteria and most maintenance and resource acquisition strategists belonging to the phylum Actinobacteria. By accounting for extracellular enzyme investment, our proposed life history strategies complement existing frameworks, such as the copiotroph-oligotroph continuum and Grime’s competitorstress tolerator-ruderal triangle. Our results have biogeochemical implications because allocation to extracellular enzymes versus growth or stress tolerance can determine the fate and form of organic matter cycling through surface soil.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Decarboxylation of the Catalytic Lysine Residue by the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the OXA-58 Carbapenemase

Antibiotic resistance in bacteria is a major global health concern. The wide spread of carbapenemases, bacterial enzymes that degrade the last-resort carbapenem antibiotics, is responsible for multidrug resistance in bacterial pathogens and has further significantly exacerbated this problem. Acinetobacter baumannii is one of the leading nosocomial pathogens due to the acquisition and wide dissemination of carbapenem-hydrolyzing class D β-lactamases, which have dramatically diminished available therapeutic options. Thus, new antibiotics that are active against multidrug-resistantA. baumannii and carbapenemase inhibitors are urgently needed. Here we report characterization of the interaction of the C5α-methyl-substituted carbapenem NA-1-157 with one of the clinically important class D carbapenemases, OXA-58. Antibiotic susceptibility testing shows that the compound is more potent than commercial carbapenems against OXA-58-producingA. baumannii, with a clinically sensitive MIC value of 1 μg/mL. Kinetic studies demonstrate that NA-1-157 is a very poor substrate of the enzyme due mainly to a significantly reduced deacylation rate. Mass spectrometry analysis shows that inhibition of OXA-58 by NA-1-157 proceeds through both the classical acyl-enzyme intermediate and a reversible covalent species. Time-resolved X-ray crystallographic studies reveal that upon acylation of the enzyme, the compound causes progressive decarboxylation of the catalytic lysine residue, thus severely impairing deacylation. Overall, this study demonstrates that the carbapenem NA-1-157 is highly resistant to degradation by the OXA-58 carbapenemase.

Acinetobactor↗

DOE OSTI2023

The genome-scale metabolic model for the purple non-sulfur bacterium Rhodopseudomonas palustris Bis A53 accurately predicts phenotypes under chemoheterotrophic, chemoautotrophic, photoheterotrophic, and photoautotrophic growth conditions

The purple non-sulfur bacterium Rhodopseudomonas palustris is recognized as a critical microorganism in the nitrogen and carbon cycle and one of the most common members in wastewater treatment communities. This bacterium is metabolically extremely versatile. It is capable of heterotrophic growth under aerobic and anaerobic conditions, but also able to grow photoautotrophically as well as mixotrophically. Therefore R . palustris can adapt to multiple environments and establish commensal relationships with other organisms, expressing various enzymes supporting degradation of amino acids, carbohydrates, nucleotides, and complex polymers. Moreover, R . palustris can degrade a wide range of pollutants under anaerobic conditions, e.g., aromatic compounds such as benzoate and caffeate, enabling it to thrive in chemically contaminated environments. However, many metabolic mechanisms employed by R . palustris to breakdown and assimilate different carbon and nitrogen sources under chemoheterotrophic or photoheterotrophic conditions remain unknown. Systems biology approaches, such as metabolic modeling, have been employed extensively to unravel complex mechanisms of metabolism. Previously, metabolic models have been reconstructed to study selected capabilities of R . palustris under limited experimental conditions. Here, we developed a comprehensive metabolic model (M-model) for R . palustris Bis A53 ( i DT1294) consisting of 2,721 reactions, 2,123 metabolites, and comprising 1,294 genes. We validated the model using high-throughput phenotypic, physiological, and kinetic data, testing over 350 growth conditions. i DT1294 achieved a prediction accuracy of 90% for growth with various carbon and nitrogen sources and close to 80% for assimilation of aromatic compounds. Moreover, the M-model accurately predicts dynamic changes of growth and substrate consumption rates over time under nine chemoheterotrophic conditions and demonstrated high precision in predicting metabolic changes between photoheterotrophic and photoautotrophic conditions. This comprehensive M-model will help to elucidate metabolic processes associated with the assimilation of multiple carbon and nitrogen sources, anoxygenic photosynthesis, aromatic compound degradation, as well as production of molecular hydrogen and polyhydroxybutyrate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Root exudation links root traits to soil functioning in agroecosystems

Root exudation is a key process for plant nutrient acquisition, but the controls on root exudation and its relationship to soil C and N processes in agroecosystems are unclear. We hypothesized that root exudation rates would be related to root morphological traits, N fertilization, and soil moisture. We also anticipated that root exudation would be correlated with bulk soil enzyme activity. Root exudation, root traits, and bulk soil extracellular enzyme activity were assessed in maize (Zea mays L.), soybean (Glycine max (L.) Merr.), biomass sorghum (Sorghum bicolor (L.) Moench), giant miscanthus (Miscanthus × giganteus), and switchgrass (Panicum virgatum L.). Measurements were taken in situ during two growing seasons with contrasting precipitation regimes, and N fertilization rate was varied in sorghum during one year. Specific root exudation (per unit root surface area) was negatively related to root diameter and was generally higher in annuals than perennials. Sorghum N fertilization did not affect root exudation rates, and soil moisture regime had no effect on annual root exudation rates within maize, sorghum, and miscanthus. Specific root exudation was negatively related to bulk soil C- and N-degrading soil enzyme activities. Intrinsic plant characteristics appeared more important than environmental variables in controlling in situ root exudation rates. The relationships between root diameter, root exudation, and soil C and N processes link root morphological traits to soil functions and demonstrate the potential tradeoffs among plant nutrient acquisition strategies in agroecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Data for Root Exudation Links Root Traits to Soil Functioning in Agroecosystems

Biomass Analytics↗

DOE OSTI2020

Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host–pathogen conflict

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Low-intensity frequent fires in coniferous forests transform soil organic matter in ways that may offset ecosystem carbon losses

The impact of shifting disturbance regimes on soil carbon (C) storage is a key uncertainty in global change research. Wildfires in coniferous forests are becoming more frequent in many regions, potentially causing large C emissions. Repeated low-intensity prescribed fires can mitigate wildfire severity, but repeated combustion may decrease soil C unless compensatory responses stabilize soil organic matter. Here, we tested how 30 years of decadal prescribed burning affected C and nitrogen (N) in plants, detritus, and soils in coniferous forests in the Sierra Nevada mountains, USA. Tree basal area and litter stocks were resilient to fire, but fire reduced forest floor C by 77% (-36.4 Mg C/ha). In mineral soils, fire reduced C that was free from minerals by 41% (-4.4 Mg C/ha) but not C associated with minerals, and only in depths ≤ 5 cm. Fire also transformed the properties of remaining mineral soil organic matter by increasing the proportion of C in a pyrogenic form (from 3.2% to 7.5%) and associated with minerals (from 46% to 58%), suggesting the remaining soil C is more resistant to decomposition. Laboratory assays illustrated that fire reduced microbial CO2 respiration rates by 55% and the activity of eight extracellular enzymes that degrade cellulosic and aromatic compounds by 40–66%. Lower decomposition was correlated with lower inorganic N (-49%), especially ammonium, suggesting N availability is coupled with decomposition. The relative increase in forms of soil organic matter that are resistant to decay or stabilized onto mineral surfaces, and the associated decline in decomposition suggest that low-intensity fires may promote mineral soil C storage in pools with long mean residence times in coniferous forests.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Review of advances in the development of laccases for the valorization of lignin to enable the production of lignocellulosic biofuels and bioproducts

Not Available

09 BIOMASS FUELS↗

DOE OSTI2022

Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Efficient utilization of agro-industrial waste, such as sugar beet pulp, is crucial for the bio-based economy. The fungus Aspergillus niger possesses a wide array of enzymes that degrade complex plant biomass substrates, and several regulators have been reported to play a role in their production. The role of the regulators GaaR, AraR, and RhaR in sugar beet pectin degradation has previously been reported. However, genetic regulation of the degradation of sugar beet pulp has not been assessed in detail. In this study, we generated a set of single and combinatorial deletion mutants targeting the pectinolytic regulators GaaR, AraR, RhaR, and GalX as well as the (hemi-)cellulolytic regulators XlnR and ClrB to address their relative contribution to the utilization of sugar beet pulp. We show that A. niger has a flexible regulatory network, adapting to the utilization of (hemi-)cellulose at early timepoints when pectin degradation is impaired.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past

Abstract Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle

Abstract With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l - and d -amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC 50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

42 ENGINEERING↗

DOE OSTI2025

Genomic factors limiting the diversity of Saccharomycotina plant pathogens

The Saccharomycotina fungi have evolved to inhabit a vast diversity of habitats over their 400-million-year evolution. There are, however, only a few known fungal pathogens of plants in this subphylum, primarily belonging to the genera Eremothecium and Geotrichum. We compared the genomes of 12 plant-pathogenic Saccharomycotina strains to 360 plant-associated strains to identify features unique to the phytopathogens. Characterization of the oxylipin synthesis genes, a compound believed to be involved in Eremothecium pathogenicity, did not reveal any differences in gene presence within or between the plant-pathogenic and plant-associated strains. A reverse-ecological approach, however, revealed that plant pathogens lack several metabolic enzymes known to assist other phytopathogens in overcoming plant defenses. This includes L-rhamnose metabolism, formamidase and nitrilase genes. This result suggests that the Saccharomycotina plant pathogens are limited to infecting ripening fruits as they are without the necessary enzymes to degrade common phytohormones and secondary metabolites produced by plants.

Saccharomycotina, fungi, phytopathogen, reverse ec↗

DOE OSTI2024

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Microbial responses to long-term warming differ across soil microenvironments

Soil carbon loss is likely to increase due to climate warming, but microbiomes and microenvironments may dampen this effect. In a 30-year warming experiment, physical protection within soil aggregates affected the thermal responses of soil microbiomes and carbon dynamics. In this study, we combined metagenomic analysis with physical characterization of soil aggregates to explore mechanisms by which microbial communities respond to climate warming across different soil microenvironments. Long-term warming decreased the relative abundances of genes involved in degrading labile compounds (e.g. cellulose), but increased those genes involved in degrading recalcitrant compounds (e.g. lignin) across aggregate sizes. These changes were observed in most phyla of bacteria, especially for Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, and Planctomycetes. Microbial community composition was considerably altered by warming, leading to declined diversity for bacteria and fungi but not for archaea. Microbial functional genes, diversity, and community composition differed between macroaggregates and microaggregates, indicating the essential role of physical protection in controlling microbial community dynamics. Our findings suggest that microbes have the capacity to employ various strategies to acclimate or adapt to climate change (e.g. warming, heat stress) by shifting functional gene abundances and community structures in varying microenvironments, as regulated by soil physical protection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Efficient and Cost-Effective Enzymatic Conversion of Lignocellulose Feedstock to Useful Bioproducts

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Enzymatic deconstruction of lignocellulose↗

DOE OSTI2021

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These important enzymes are often bimodular, including a catalytic domain and carbohydrate binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able discriminate GH7 CBHs and EGs with up to 99% accuracy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our ML results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

Abstract Background Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei , as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

09 BIOMASS FUELS↗