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DOE OSTI2021

The Azospirillum brasilense Core Chemotaxis Proteins CheA1 and CheA4 Link Chemotaxis Signaling with Nitrogen Metabolism

Bacterial chemotaxis affords motile bacteria the ability to navigate the environment to locate niches for growth and survival. At the molecular level, chemotaxis depends on chemoreceptor signaling arrays that interact with cytoplasmic proteins to control the direction of movement. In Azospirillum brasilense , chemotaxis is mediated by two distinct chemotaxis pathways: Che1 and Che4. Both Che1 and Che4 are critical in the A. brasilense free-living and plant-associated lifestyles. Here, we use whole-cell proteomics and metabolomics to characterize the role of chemotaxis in A. brasilense physiology. We found that mutants lacking CheA1 or CheA4 or both are affected in nonchemotaxis functions, including major changes in transcription, signaling transport, and cell metabolism. We identify specific effects of CheA1 and CheA4 on nitrogen metabolism, including nitrate assimilation and nitrogen fixation, that may depend, at least, on the transcriptional control of rpoN , which encodes RpoN, a global regulator of metabolism, including nitrogen. Consistent with proteomics, the abundance of several nitrogenous compounds (purines, pyrimidines, and amino acids) changed in the metabolomes of the chemotaxis mutants relative to the parental strain. Further, we uncover novel, and yet uncharacterized, layers of transcriptional and posttranscriptional control of nitrogen metabolism regulators. Together, our data reveal roles for CheA1 and CheA4 in linking chemotaxis and nitrogen metabolism, likely through control of global regulatory networks.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Changes in carbon and nitrogen metabolism during seawater-induced mortality of Picea sitchensis trees

Abstract Increasing seawater exposure is causing mortality of coastal forests, yet the physiological response associated with seawater-induced tree mortality, particularly in non-halophytes, is poorly understood. We investigated the shifts in carbon and nitrogen (N) metabolism of mature Sitka-spruce trees that were dying after an ecosystem-scale manipulation of tidal seawater exposure. Soil porewater salinity and foliar ion concentrations increased after seawater exposure and were strongly correlated with the percentage of live foliated crown (PLFC; e.g., crown ‘greenness’, a measure of progression to death). Co-occurring with decreasing PLFC was decreasing photosynthetic capacity, N-investment into photosynthesis, N-resorption efficiency and non-structural carbohydrate (soluble sugars and starch) concentrations, with the starch reserves depleted to near zero when PLFC dropped below 5%. Combined with declining PLFC, these changes subsequently decreased total carbon gain and thus exacerbated the carbon starvation process. This study suggests that an impairment in carbon and N metabolism during the mortality process after seawater exposure is associated with the process of carbon starvation, and provides critical knowledge necessary to predict sea-level rise impacts on coastal forests.

Li, Weibin↗

DOE OSTI2022

Genome Streamlining, Proteorhodopsin, and Organic Nitrogen Metabolism in Freshwater Nitrifiers

Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth’s largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) “Candidatus Nitrotoga” and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Nitrogen Metabolism in Pseudomonas putida : Functional Analysis Using Random Barcode Transposon Sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. So we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Dominant nitrogen metabolisms of a warm, seasonally anoxic freshwater ecosystem revealed using genome resolved metatranscriptomics

In aquatic ecosystems where primary productivity is limited by nitrogen (N), whether continuously, seasonally, or in concert with additional nutrient limitations, increased inorganic N availability can reshape ecosystem structure and function, potentially resulting in eutrophication and even harmful algal blooms. Whereas microbial metabolic processes such as mineralization and dissimilatory nitrate reduction to ammonium increase inorganic N availability, denitrification removes bioavailable N from the ecosystem. Therefore, understanding these key microbial mechanisms is critical to the sustainable management and environmental stewardship of inland freshwater resources. This study identifies and characterizes these crucial metabolisms in a warm, seasonally anoxic ecosystem. Results are contextualized by an ecological understanding of the study system derived from a multi-year continuous monitoring effort. This unique data set is the first of its kind in this largely understudied ecosystem (tropical lakes) and also provides insight into microbiome function and associated taxa in warm, anoxic freshwaters.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Microbiome engineering for sustainable agriculture: using synthetic biology to enhance nitrogen metabolism in plant-associated microbes

Plants benefit from symbiotic relationships with their microbiomes. Modifying these microbiomes to further promote plant growth and improve stress tolerance in crops is a promising strategy. However, such efforts have had limited success, perhaps because the original microbiomes quickly re-establish. Since the complex biological networks involved are little understood, progress through conventional means is time-consuming. Synthetic biology, with its practical successes in multiple industries, could speed up this research considerably. Some fascinating candidates for production by synthetic microbiomes are organic nitrogen metabolites and related pyridoxal-5'-phosphate-dependent enzymes, which have pivotal roles in microbe-microbe and plant-microbe interactions. This review summarizes recent studies of these metabolites and enzymes and discusses prospective synthetic biology platforms for sustainable agriculture.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

PII interactions with the acetyl-CoA carboxylase subunits BADC and BCCP co-regulate lipid and nitrogen metabolism in Arabidopsis

In plants, the initiation of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACCase), which produces malonyl-CoA. The heteromeric form of ACCase (htACCase) is a holoenzyme consisting of biotin carboxylase and carboxyltransferase sub-complexes, both of which are subject to extensive regulation. Biotin carboxylase activity is controlled in part by the presence of the catalytic biotin carboxyl carrier proteins (BCCP1/2) and/or the non-catalytic, non-biotinylated, biotin/lipoyl attachment domain-containing proteins (BADC1/2/3) that associate with backbone biotin carboxylase (BC) protein. However, the mechanisms regulating BADC and BCCP interactions with BC and, consequently, ACCase activity in planta , remain unclear. Here, we demonstrate that the Arabidopsis ( Arabidopsis thaliana ) regulatory protein PII modulates htACCase activity through independent interactions with BADC and BCCP proteins in a selective manner. Analysis of badc1 badc2 and badc1 badc3 mutant lines and the respective pii triple mutants revealed that changes in seed oil and protein accumulation of badc double mutants are PII/nitrogen dependent. Absolute quantification of htACCase subunits and PII in developing seeds suggests that Arabidopsis exerts tight regulation over individual protein stoichiometry to balance oil and protein accumulation. The effects on vegetative and seed development indicate that PII and BADC proteins have distinct yet overlapping roles in regulating plant metabolism.

Garneau, Matthew G. [Washington State Univ., Pullm↗

DOE OSTI2024

Genomic factors shape carbon and nitrogen metabolic niche breadth across Saccharomycotina yeasts

Organisms exhibit extensive variation in ecological niche breadth, from very narrow (specialists) to very broad (generalists). Two general paradigms have been proposed to explain this variation: (i) trade-offs between performance efficiency and breadth and (ii) the joint influence of extrinsic (environmental) and intrinsic (genomic) factors. We assembled genomic, metabolic, and ecological data from nearly all known species of the ancient fungal subphylum Saccharomycotina (1154 yeast strains from 1051 species), grown in 24 different environmental conditions, to examine niche breadth evolution. We found that large differences in the breadth of carbon utilization traits between yeasts stem from intrinsic differences in genes encoding specific metabolic pathways, but we found limited evidence for trade-offs. Furthermore, these comprehensive data argue that intrinsic factors shape niche breadth variation in microbes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Integrative analysis of CAM photosynthesis reveals its impact on primary metabolism in Yucca

Crassulacean Acid Metabolism (CAM) is an adaptation that temporally separates carbon uptake at night from photosynthesis during the day. CAM has evolved repeatedly across vascular plants, as its emergence may depend on simple regulatory changes to deeply conserved metabolic pathways. Modern CAM research relies heavily on interpretation of transcriptomic data, though regulation occurs at multiple levels following transcription. Additionally, while most research to date has focused on a handful of genes and metabolites in the core CAM pathway, the co-option of conserved regulatory and functional genes is bound to have wide-ranging effects on other aspects of primary metabolism. In this study, we integrate transcriptomic, proteomic, and metabolomic data to compare primary metabolism between the CAM species Yucca aloifolia and closely related C 3 species, Y. filamentosa. We observe minimal correlation between protein abundance and mRNA expression, suggesting significant post-transcriptional regulation in CAM species. We also find evidence of shifts in gene expression and metabolite accumulation outside of the central CAM pathway, suggesting that the shift to CAM has cascading effects across primary metabolism, especially nitrogen metabolism. Our findings provide insights into the metabolic shifts associated with CAM evolution, and highlight the complexity of its regulation at multiple biological levels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis

Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol.

09 BIOMASS FUELS↗

DOE OSTI2022

Integrated flux and pool size analysis in plant central metabolism reveals unique roles of glycine and serine during photorespiration

Photorespiration is an essential process juxtaposed between plant carbon and nitrogen metabolism that responds to dynamic environments. Photorespiration recycles inhibitory intermediates arising from oxygenation reactions catalysed by Rubisco back into the C 3 cycle, but it is unclear what proportions of its nitrogen-containing intermediates (glycine and serine) are exported into other metabolisms in vivo and how these pool sizes affect net CO 2 gas exchange during photorespiratory transients. Here, to address this uncertainty, we measured rates of amino acid export from photorespiration using isotopically non-stationary metabolic flux analysis. This analysis revealed that ~23–41% of the photorespiratory carbon was exported from the pathway as serine under various photorespiratory conditions. Furthermore, we determined that the build-up and relaxation of glycine pools constrained a large portion of photosynthetic acclimation during photorespiratory transients. Furthermore, these results reveal the unique and important roles of glycine and serine in successfully maintaining various photorespiratory fluxes that occur under environmental fluctuations in nature and providing carbon and nitrogen for metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440

ABSTRACT There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida . Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

09 BIOMASS FUELS↗

DOE OSTI2023

Taxogenomic analysis of a novel yeast species isolated from soil, Pichia galeolata sp. nov.

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446T (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

SRX13165144: yHMH446_Pichia_sp_nov_plate33_SPADES

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2022

Pichia sp. yHMH446, whole genome shotgun sequencing project

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2021

Pichia sp. strain yHMH446 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2021

Pichia sp. strain yHMH446 internal transcribed spacer 2 and large subunit ribosomal RNA gene, partial sequence

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗