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.

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.

Syntrophic splitting of central carbon metabolism in host cells bearing functionally different symbiotic bacteria.

Insects feeding on the nutrient-poor diet of xylem plant sap generally bear two microbial symbionts that are localized to different organs (bacteriomes) and provide complementary sets of essential amino acids (EAAs). Here, we investigate the metabolic basis for the apparent paradox that xylem-feeding insects are under intense selection for metabolic efficiency but incur the cost of maintaining two symbionts for functions mediated by one symbiont in other associations. Using stable isotope analysis of central carbon metabolism and metabolic modeling, we provide evidence that the bacteriomes of the spittlebug Clastoptera proteus display high rates of aerobic glycolysis, with syntrophic splitting of glucose oxidation. Specifically, our data suggest that one bacteriome (containing the bacterium Sulcia, which synthesizes seven EAAs) predominantly processes glucose glycolytically, producing pyruvate and lactate, and the exported pyruvate and lactate is assimilated by the second bacteriome (containing the bacterium Zinderia, which synthesizes three energetically costly EAAs) and channeled through the TCA cycle for energy generation by oxidative phosphorylation. We, furthermore, calculate that this metabolic arrangement supports the high ATP demand in Zinderia bacteriomes for Zinderia-mediated synthesis of energy-intensive EAAs. We predict that metabolite cross-feeding among host cells may be widespread in animal-microbe symbioses utilizing low-nutrient diets.

Advancements in 13C isotope tracking of synergistic substrate co-utilization in Pseudomonas species and implications for biotechnology applications.

Pseudomonads are well-known to thrive in diverse and complex nutritional habitats, and these capabilities make Pseudomonas species attractive as whole-cell biocatalysts. Industrial bioconversion processes often rely on complex uptake and synergistic metabolic systems due to the presence of varied carbon substrates in nutrient feedstocks. Isotope labeling experiments (ILEs) are emerging techniques used to elucidate cell metabolism following feeding on isotopically enriched substrates and are pivotal to the understanding of carbon partitioning during co-utilization. In this review, we highlight the applications of ILEs to decipher the metabolic networks in Pseudomonas species and evaluate their relevance in optimizing biocatalytic platforms.

The Metabolome of Associations between Xylem-Feeding Insects and their Bacterial Symbionts.

Metabolomics has increasingly led to important insights in chemical ecology by identifying environmentally relevant small molecules that mediate inter-organismal interactions. Nevertheless, the application of metabolomics to investigate interactions between phytophagous insects and their microbial symbionts remains underutilized. Here, we investigated the metabolomes of the bacteriomes (organs bearing symbiotic bacteria) isolated from natural populations of five species of xylem-feeding insects. We identified three patterns. First, the metabolomes varied among the five species, likely influenced by insect phylogeny, food plant and taxonomic identity of the symbionts. Second, the ratio of glutamine: glutamate in the bacteriomes was 0.7-3.6 to 1, indicative of nitrogen-sufficient metabolism and raising the possibility that the insect sustains nitrogen-enriched status of the bacteriomes despite the nitrogen scarcity of the xylem diet. Finally, bacteriomes from insect species bearing genetically-similar symbionts displayed limited variation in their metabolomes, suggesting that the metabolic pattern of the bacteriome metabolic pools is correlated with the genetic repertoire of the symbionts. Altogether, these metabolomic patterns yield specific hypotheses of underlying processes that are testable by wider sampling of natural populations and experimental study.

Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida.

Pseudomonas species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling Pseudomonas to utilize mixed substrates remain poorly understood. Here, we employed a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in Pseudomonas putida KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively a monomer of carbohydrate polymers and a derivative of lignin monomers. Despite nearly equal consumption of both substrates, metabolite isotopologues revealed nonuniform assimilation throughout the metabolic network. Gluconeogenic flux of benzoate-derived carbons from the tricarboxylic acid cycle did not reach the upper Embden-Meyerhof-Parnas pathway nor the pentose-phosphate pathway. These latter two pathways were populated exclusively by glucose-derived carbons through a cyclic connection with the Entner-Doudoroff pathway. We integrated the 13C-metabolomics data with physiological parameters for quantitative flux analysis, demonstrating that the metabolic segregation of the substrate carbons optimally sustained biosynthetic flux demands and redox balance. Changes in protein abundance partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture versus on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes. These findings provide new insights into the metabolic architecture that affords adaptability of P. putida to divergent carbon substrates and highlight regulatory points at different metabolic nodes that may underlie the high nutritional flexibility of Pseudomonas species.

A Cyclic Metabolic Network in Pseudomonas protegens Pf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization.

The genetic characterization of Pseudomonas protegens Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed 13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes in P. protegens Pf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates in P. protegens Pf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCE Species of the Pseudomonas genus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism in Pseudomonas protegens Pf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network in P. protegens is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped Pseudomonas species.

Quantitation of carbohydrate monomers and dimers by liquid chromatography coupled with high-resolution mass spectrometry.

As remnants of plant wastes or plant secretions, carbohydrates are widely found in various environmental matrices. Carbohydrate-containing feedstocks represent important carbon sources for engineered bioproduction of commodity compounds. Routine monitoring and quantitation of heterogenous carbohydrate mixtures requires fast, accurate, and precise analytical methods. Here we present two methods to quantify carbohydrates mixtures by coupling hydrophilic interaction liquid chromatography with electrospray ionization high-resolution mass spectrometry. Method 1 was optimized for eleven different carbohydrates: three pentoses (ribose, arabinose, xylose), three hexoses (glucose, fructose, mannose), and five dimers (sucrose, cellobiose, maltose, trehalose, lactose). Method 1 can monitor these carbohydrates simultaneously, except in the case of co-elution of xylose/arabinose and lactose/maltose/cellobiose peaks. Using the same stationary and mobile phases as in Method 1, Method 2 was developed to separate glucose and galactose, which were indistinguishable in Method 1. Both methods have low limits of detection (0.019-0.40 µM) and quantification (0.090-1.3 µM), good precision (2.4-13%) except sucrose (18%), and low mass error (0.0-2.4 ppm). Method 1 was robust at analyzing high ionic strength solutions, but a moderate matrix effect was observed. Finally, we apply Method 1 to track concurrently the extracellular depletion of five carbohydrates (xylose, glucose, fructose, mannose, and maltose) by Pseudomonas protegens Pf-5, a biotechnologically-important soil bacterial species.