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Abstract 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 13 C 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. 

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.

 

Inorganic pyrophosphatase (PPase) is an enzyme that catalyzes the hydrolysis of the phosphoanhydride bond in pyrophosphate (PPi) to release inorganic phosphate (Pi) and simultaneously exchange oxygen isotopes between Pi and water. Here, we quantified the exchange kinetics of oxygen isotopes between five Pi isotopologues (P18O4, P18O316O, P18O216O2, P18O16O3, and P16O4) and water using Raman spectroscopy and 31P nuclear magnetic resonance (NMR) during the PPase-catalyzed 18O–16O isotope exchange reaction in Pi-water and PPi-water systems. At a high PPi concentration (300 mM), hydrolysis of PPi by PPase was predominant, and only a small fraction of PPi (≪1%) took part in the reversible hydrolysis–condensation reaction (PPi ↔ Pi), leading to the oxygen isotope exchange between Pi and water. We demonstrated that Raman and NMR methods can be equally applied for monitoring the kinetics of the oxygen exchange between the Pi isotopologue and water. It was found that the isotope exchange determined by the spectroscopic methods was detectable as low as 0.2% 18O abundance, but the reliability below 1% was much lower. Given that high P concentrations (≥1 mM) are required in these methods, environmental application of these methods is limited to rare high P conditions in engineered and agricultural environments. 

ABSTRACT 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 13 C-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. 

ABSTRACT 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. 

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 13 C-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.