Disproportionate Carbon Dioxide Efflux in Bacterial Metabolic Pathways for Different Organic Substrates Leads to Variable Contribution to Carbon-Use Efficiency.

Microbial organic matter turnover is an important contributor to the terrestrial carbon dioxide (CO2) budget. Partitioning of organic carbons into biomass relative to CO2 efflux, termed carbon-use efficiency (CUE), is widely used to characterize organic carbon cycling by soil microorganisms. Recent studies challenge proposals of CUE dependence on the oxidation state of the substrate carbon and implicate instead metabolic strategies. Still unknown are the metabolic mechanisms underlying variability in CUE. We performed a multiomics investigation of these mechanisms in Pseudomonas putida, a versatile soil bacterium of the Gammaproteobacteria, processing a mixture of plant matter derivatives. Our 13C-metabolomics data captured substrate carbons into different metabolic pathways: cellulose-derived sugar carbons in glycolytic and pentose-phosphate pathways; lignin-related aromatic carbons in the tricarboxylic acid cycle. Subsequent 13C-metabolic flux analysis revealed a 3-fold lower investment of sugar carbons in CO2 efflux compared to aromatic carbons, in agreement with reported substrate-dependent CUE. Proteomics analysis revealed enzyme-level regulation only for substrate uptake and initial catabolism, which dictated downstream fluxes through CO2-producing versus biomass-synthesizing reactions. Metabolic partitioning as shown here explained the substrate-dependent CUE calculated from reported metabolic flux analyses of other bacteria, further supporting a metabolism-guided perspective for predicting the microbial conversion of accessible organic matter to CO2 efflux.

Deconstructing synthetic biology across scales: a conceptual approach for training synthetic biologists.

Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales-molecular, circuit/network, cell/cell-free systems, biological communities, and societal-giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists.

Electrostatic coupling and water bridging in adsorption hierarchy of biomolecules at water-clay interfaces.

Clay minerals are implicated in the retention of biomolecules within organic matter in many soil environments. Spectroscopic studies have proposed several mechanisms for biomolecule adsorption on clays. Here, we employ molecular dynamics simulations to investigate these mechanisms in hydrated adsorbate conformations of montmorillonite, a smectite-type clay, with ten biomolecules of varying chemistry and structure, including sugars related to cellulose and hemicellulose, lignin-related phenolic acid, and amino acids with different functional groups. Our molecular modeling captures biomolecule-clay and biomolecule-biomolecule interactions that dictate selectivity and competition in adsorption retention and interlayer nanopore trapping, which we determine experimentally by NMR and X-ray diffraction, respectively. Specific adsorbate structures are important in facilitating the electrostatic attraction and Van der Waals energies underlying the hierarchy in biomolecule adsorption. Stabilized by a network of direct and water-bridged hydrogen bonds, favorable electrostatic interactions drive this hierarchy whereby amino acids with positively charged side chains are preferentially adsorbed on the negatively charged clay surface compared to the sugars and carboxylate-rich aromatics and amino acids. With divalent metal cations, our model adsorbate conformations illustrate hydrated metal cation bridging of carboxylate-containing biomolecules to the clay surface, thus explaining divalent cation-promoted adsorption from our experimental data. Adsorption experiments with a mixture of biomolecules reveal selective inhibition in biomolecule adsorption, which our molecular modeling attributes to electrostatic biomolecule-biomolecule pairing that is more energetically favorable than the biomolecule-clay complex. In sum, our findings highlight chemical and structural features that can inform hypotheses for predicting biomolecule adsorption at water-clay interfaces.

Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation.

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter.

Methanogenesis allows methanogenic archaea to generate cellular energy for their growth while producing methane. Thermophilic hydrogenotrophic species of the genus Methanothermobacter have been recognized as robust biocatalysts for a circular carbon economy and are already applied in power-to-gas technology with biomethanation, which is a platform to store renewable energy and utilize captured carbon dioxide. Here, we generated curated genome-scale metabolic reconstructions for three Methanothermobacter strains and investigated differences in the growth performance of these same strains in chemostat bioreactor experiments with hydrogen and carbon dioxide or formate as substrates. Using an integrated systems biology approach, we identified differences in formate anabolism between the strains and revealed that formate anabolism influences the diversion of carbon between biomass and methane. This finding, together with the omics datasets and the metabolic models we generated, can be implemented for biotechnological applications of Methanothermobacter in power-to-gas technology, and as a perspective, for value-added chemical production.

Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants.

While genome sequencing has expanded our knowledge of symbiosis, role assignment within multi-species microbiomes remains challenging due to genomic redundancy and the uncertainties of in vivo impacts. We address such questions, here, for a specialized nitrogen (N) recycling microbiome of turtle ants, describing a new genus and species of gut symbiont-Ischyrobacter davidsoniae (Betaproteobacteria: Burkholderiales: Alcaligenaceae)-and its in vivo physiological context. A re-analysis of amplicon sequencing data, with precisely assigned Ischyrobacter reads, revealed a seemingly ubiquitous distribution across the turtle ant genus Cephalotes, suggesting ≥50 million years since domestication. Through new genome sequencing, we also show that divergent I. davidsoniae lineages are conserved in their uricolytic and urea-generating capacities. With phylogenetically refined definitions of Ischyrobacter and separately domesticated Burkholderiales symbionts, our FISH microscopy revealed a distinct niche for I. davidsoniae, with dense populations at the anterior ileum. Being positioned at the site of host N-waste delivery, in vivo metatranscriptomics and metabolomics further implicate I. davidsoniae within a symbiont-autonomous N-recycling pathway. While encoding much of this pathway, I. davidsoniae expressed only a subset of the requisite steps in mature adult workers, including the penultimate step deriving urea from allantoate. The remaining steps were expressed by other specialized gut symbionts. Collectively, this assemblage converts inosine, made from midgut symbionts, into urea and ammonia in the hindgut. With urea supporting host amino acid budgets and cuticle synthesis, and with the ancient nature of other active N-recyclers discovered here, I. davidsoniae emerges as a central player in a conserved and impactful, multipartite symbiosis.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism.

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Metabolomics analysis of unresolved molecular variability in stoichiometry dynamics of a stream dissolved organic matter.

Broad molecular classification based on stoichiometric ratio relationships has been used extensively to characterize the chemical diversity of aquatic dissolved organic matter (DOM). However, variability in the molecular composition within this classification has remained elusive, thus limiting the interpretation of DOM dynamics, especially with respect to transport versus transformation patterns in response to hydrologic or landscape changes. Here, leveraging high-frequency spatiotemporal sampling during rainfall events at a Critical Zone Observatory project site in Clear Creek, Iowa, we apply a metabolomics-based analysis validated with fragmentation using tandem mass spectrometry to uncover patterns in the molecular features of the DOM composition that were not resolved by classification based on stoichiometric ratios in the chemical formulae. From upstream to downstream sites, beyond the increased aromaticity implied by changes in the stoichiometric ratios, we identified an increased abundance of flavonoids and other phenylpropanoids, two important subgroups of aromatic compounds. The stoichiometric analysis also proposed a localized decline in the abundance of lipid-like compounds, which we attributed specifically to medium-chain and short-chain fatty acids; other lipids such as long-chain fatty acids and sterol lipids remained unchanged. We further determined in-stream molecular transitions and specific compound degradation by capturing changes in the molecular masses of terpenoids, phenylpropanoids, fatty acids, and amino acids. In sum, the metabolomics analysis of the chemical formulae resolved molecular variability imprinted on the stoichiometric DOM composition to implicate key molecular subgroups underlying carbon transport and cycling dynamics in the stream.

Dynamic utilization of low-molecular-weight organic substrates across a microbial growth rate gradient.

AIM: Low-molecular-weight organic substances (LMWOSs) are at the nexus between micro-organisms, plant roots, detritus, and the soil mineral matrix. The nominal oxidation state of carbon (NOSC) has been suggested as a potential parameter for modelling microbial uptake rates of LMWOSs and the efficiency of carbon incorporation into new biomass. METHODS AND RESULTS: In this study, we assessed the role of compound class and oxidation state on uptake kinetics and substrate-specific carbon use efficiency (SUE) during the growth of three model soil micro-organisms, a fungal isolate (Penicillium spinulosum) and two bacterial isolates (Paraburkholderia solitsugae, and Ralstonia pickettii). Isolates were chosen that spanned a growth rate gradient (0.046-0.316 h-1 ) in media containing 34 common LMWOSs at realistically low initial concentrations (25 µM each). Clustered, co-utilization of LMWOSs occurred for all three organisms. Potential trends (p < 0.05) for early utilization of more oxidized substrates were present for the two bacterial isolates (P. solitsugae and R. pickettii), but high variability (R2 < 0.15) and a small effect of NOSC indicate these relationships are not useful for prediction. The SUEs of selected substrates ranged from 0.16 to 0.99 and there was no observed relationship between NOSC and SUE. CONCLUSION: Our results do not provide compelling population-level support for NOSC as a predictive tool for either uptake kinetics or the efficiency of use of LMWOS in soil solution. SIGNIFICANCE AND IMPACT OF THE STUDY: Metabolic strategies of organisms are likely more important than chemical identity in determining LMWOS cycling in soils. Previous community-level observations may be biased towards fast-responding bacterial community members.

Strategies of organic phosphorus recycling by soil bacteria: acquisition, metabolism, and regulation.

Critical to meeting cellular phosphorus (P) demand, soil bacteria deploy a number of strategies to overcome limitation in inorganic P (Pi ) in soils. As a significant contributor to P recycling, soil bacteria secrete extracellular enzymes to degrade organic P (Po ) in soils into the readily bioavailable Pi . In addition, several Po compounds can be transported directly via specific transporters and subsequently enter intracellular metabolic pathways. In this review, we highlight the strategies that soil bacteria employ to recycle Po from the soil environment. We discuss the diversity of extracellular phosphatases in soils, the selectivity of these enzymes towards various Po biomolecules and the influence of the soil environmental conditions on the enzyme's activities. Moreover, we outline the intracellular metabolic pathways for Po biosynthesis and transporter-assisted Po and Pi uptake at different Pi availabilities. We further highlight the regulatory mechanisms that govern the production of phosphatases, the expression of Po transporters and the key metabolic changes in P metabolism in response to environmental Pi availability. Due to the depletion of natural resources for Pi , we propose future studies needed to leverage bacteria-mediated P recycling from the large pools of Po in soils or organic wastes to benefit agricultural productivity.

Effects of Phosphonate Herbicides on the Secretions of Plant-Beneficial Compounds by Two Plant Growth-Promoting Soil Bacteria: A Metabolomics Investigation.

Plant growth-promoting rhizobacteria (PGPR) that colonize plant roots produce a variety of plant-beneficial compounds, including plant-growth regulators, metal-scavenging compounds, and antibiotics against plant pathogens. Adverse effects of phosphonate herbicides, the most extensively used herbicides, on the growth and metabolism of PGPR species have been widely reported. However, the potential consequence of these effects on the biosynthesis and secretion of PGPR-derived beneficial compounds still remains to be investigated. Here, using high-resolution mass spectrometry and a metabolomics approach, we investigated both the intracellular metabolome and the extracellular secretions of biomass-normalized metabolite levels in two PGPR species (Pseudomonas protegens Pf-5, a Gram-negative bacterium; Priestia megaterium QM B1551, a Gram-positive bacterium) exposed to three common phosphonate herbicides (glyphosate, glufosinate, and fosamine; 0.1-1 mM) in either iron (Fe)-replete or Fe-deficient nutrient media. We quantified secreted auxin-type plant hormone compounds (phenylacetic acid and indole-3-acetic acid), iron-scavenging compounds or siderophores (pyoverdine and schizokinen), and antibiotics (2,4-diacetylphloroglucinol and pyoluteorin) produced by these PGPR species. The Fe-replete cells exposed to the phosphonate herbicides yielded up to a 25-fold increase in the production of both auxin and antibiotic compounds, indicating that herbicide exposure under Fe-replete conditions triggered metabolite secretions. However, the herbicide-exposed Fe-deficient cells exhibited a near 2-fold depletion in the secretion of these auxin and antibiotic compounds as well as a 77% decrease in siderophore production. Intracellular metabolomics analysis of the Fe-deficient cells further revealed metabolic perturbations in biosynthetic pathways consistent with the impaired production of the plant-beneficial compounds. Our findings implied that compromised cellular metabolism during nutrient deficiency may exacerbate the adverse effects of phosphonate herbicides on PGPR species.

Analogous Metabolic Decoupling in Pseudomonas putida and Comamonas testosteroni Implies Energetic Bypass to Facilitate Gluconeogenic Growth.

Gluconeogenic carbon metabolism is not well understood, especially within the context of flux partitioning between energy generation and biomass production, despite the importance of gluconeogenic carbon substrates in natural and engineered carbon processing. Here, using multiple omics approaches, we elucidate the metabolic mechanisms that facilitate gluconeogenic fast-growth phenotypes in Pseudomonas putida and Comamonas testosteroni, two Proteobacteria species with distinct metabolic networks. In contrast to the genetic constraint of C. testosteroni, which lacks the enzymes required for both sugar uptake and a complete oxidative pentose phosphate (PP) pathway, sugar metabolism in P. putida is known to generate surplus NADPH by relying on the oxidative PP pathway within its characteristic cyclic connection between the Entner-Doudoroff (ED) and Embden-Meyerhoff-Parnas (EMP) pathways. Remarkably, similar to the genome-based metabolic decoupling in C. testosteroni, our 13C-fluxomics reveals an inactive oxidative PP pathway and disconnected EMP and ED pathways in P. putida during gluconeogenic feeding, thus requiring transhydrogenase reactions to supply NADPH for anabolism in both species by leveraging the high tricarboxylic acid cycle flux during gluconeogenic growth. Furthermore, metabolomics and proteomics analyses of both species during gluconeogenic feeding, relative to glycolytic feeding, demonstrate a 5-fold depletion in phosphorylated metabolites and the absence of or up to a 17-fold decrease in proteins of the PP and ED pathways. Such metabolic remodeling, which is reportedly lacking in Escherichia coli exhibiting a gluconeogenic slow-growth phenotype, may serve to minimize futile carbon cycling while favoring the gluconeogenic metabolic regime in relevant proteobacterial species. IMPORTANCE Glycolytic metabolism of sugars is extensively studied in the Proteobacteria, but gluconeogenic carbon sources (e.g., organic acids, amino acids, aromatics) that feed into the tricarboxylic acid (TCA) cycle are widely reported to produce a fast-growth phenotype, particularly in species with biotechnological relevance. Much remains unknown about the importance of glycolysis-associated pathways in the metabolism of gluconeogenic carbon substrates. Here, we demonstrate that two distinct proteobacterial species, through genetic constraints or metabolic regulation at specific metabolic nodes, bypass the oxidative PP pathway during gluconeogenic growth and avoid unnecessary carbon fluxes by depleting protein investment into connected glycolysis pathways. Both species can leverage instead the high TCA cycle flux during gluconeogenic feeding to meet NADPH demand. Importantly, lack of a complete oxidative pentose phosphate pathway is a widespread metabolic trait in Proteobacteria with a gluconeogenic carbon preference, thus highlighting the important relevance of our findings toward elucidating the metabolic architecture in these bacteria.

Molecular Coordination, Structure, and Stability of Metal-Polyphosphate Complexes Resolved by Molecular Modeling and X-ray Scattering: Structural Insights on the Biological Fate of Polyphosphate.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 Å for Mg2+ but this distance was 2.64-2.70 Å for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

Hierarchical Reactivity of Enzyme-Mediated Phosphorus Recycling from Organic Mixtures by Aspergillus niger Phytase.

Biological recycling of inorganic phosphorus (Pi) from organic phosphorus (Po) compounds by phosphatase-type enzymes, including phytases, is an important contributor to the pool of bioavailable P to plants and microorganisms. However, studies of mixed-substrate reactions with these enzymes are lacking. Here, we explore the reactivity of a phytase extract from the fungus Aspergillus niger toward a heterogeneous mixture containing, in addition to phytate, different structures of environmentally relevant Po compounds such as ribonucleotides and sugar phosphates. Using a high-resolution liquid chromatography-mass spectrometry method to monitor simultaneously the parent Po compounds and their by-products, we captured sequential substrate-specific evolution of Pi from the mixture, with faster hydrolysis of multiphosphorylated compounds (phytate, diphosphorylated sugars, and di- and tri-phosphorylated ribonucleotides) than hydrolysis of monophosphorylated compounds (monophosphorylated sugars and monophosphorylated ribonucleotides). The interaction mechanisms and energies revealed by molecular docking simulations of each Po compound within the enzyme's active site explained the substrate hierarchy observed experimentally. Specifically, the favorable orientation for binding of the negatively charged phosphate moieties with respect to the positive potential surface of the active site was important. Collectively, our findings provide mechanistic insights about the broad but hierarchical role of phytase-type enzymes in Pi recycling from the heterogeneous assembly of Po compounds in agricultural soils or wastes.

Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soil Pseudomonas species.

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis-anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.

Ecophysiological Study of Paraburkholderia sp. Strain 1N under Soil Solution Conditions: Dynamic Substrate Preferences and Characterization of Carbon Use Efficiency.

We used time-resolved metabolic footprinting, an important technical approach used to monitor changes in extracellular compound concentrations during microbial growth, to study the order of substrate utilization (i.e., substrate preferences) and kinetics of a fast-growing soil isolate, Paraburkholderia sp. strain 1N. The growth of Paraburkholderia sp. 1N was monitored under aerobic conditions in a soil-extracted solubilized organic matter medium, representing a realistic diversity of available substrates and gradient of initial concentrations. We combined multiple analytical approaches to track over 150 compounds in the medium and complemented this with bulk carbon and nitrogen measurements, allowing estimates of carbon use efficiency throughout the growth curve. Targeted methods allowed the quantification of common low-molecular-weight substrates: glucose, 20 amino acids, and 9 organic acids. All targeted compounds were depleted from the medium, and depletion followed a sigmoidal curve where sufficient data were available. Substrates were utilized in at least three distinct temporal clusters as Paraburkholderia sp. 1N produced biomass at a cumulative carbon use efficiency of 0.43. The two substrates with highest initial concentrations, glucose and valine, exhibited longer usage windows, at higher biomass-normalized rates, and later in the growth curve. Contrary to hypotheses based on previous studies, we found no clear relationship between substrate nominal oxidation state of carbon (NOSC) or maximal growth rate and the order of substrate depletion. Under soil solution conditions, the growth of Paraburkholderia sp. 1N induced multiauxic substrate depletion patterns that could not be explained by the traditional paradigm of catabolite repression.IMPORTANCE Exometabolomic footprinting methods have the capability to provide time-resolved observations of the uptake and release of hundreds of compounds during microbial growth. Of particular interest is microbial phenotyping under environmentally relevant soil conditions, consisting of relatively low concentrations and modeling pulse input events. Here, we show that growth of a bacterial soil isolate, Paraburkholderia sp. 1N, on a dilute soil extract resulted in a multiauxic metabolic response, characterized by discrete temporal clusters of substrate depletion and metabolite production. Our data did not support the hypothesis that compounds with lower energy content are used preferentially, as each cluster contained compounds with a range of nominal oxidation states of carbon. These new findings with Paraburkholderia sp. 1N, which belongs to a metabolically diverse genus, provide insights on ecological strategies employed by aerobic heterotrophs competing for low-molecular-weight substrates in soil solution.