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

   https://doi.org/10.1128/mbio.03259-21  

   Wilkes, Rebecca A. ; Waldbauer, Jacob ; Aristilde, Ludmilla ( December 2021 , mBio)

   Dunn, Anne K. ; Ruby, Edward G. (Ed.)

   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 themore »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.« less
2. Proteogenomic analyses indicate bacterial methylotrophy and archaeal heterotrophy are prevalent below the grass root zone

   https://doi.org/10.7717/peerj.2687  

   Butterfield, Cristina N. ; Li, Zhou ; Andeer, Peter F. ; Spaulding, Susan ; Thomas, Brian C. ; Singh, Andrea ; Hettich, Robert L. ; Suttle, Kenwyn B. ; Probst, Alexander J. ; Tringe, Susannah G. ; et al ( January 2016 , PeerJ)

   Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO 2 . However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation.more »Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.« less
3. Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

   https://doi.org/10.1038/s41396-020-00805-w  

   Lawson, Christopher E. ; Nuijten, Guylaine H. ; de Graaf, Rob M. ; Jacobson, Tyler B. ; Pabst, Martin ; Stevenson, David. M. ; Jetten, Mike S. ; Noguera, Daniel R. ; McMahon, Katherine D. ; Amador-Noguez, Daniel ; et al ( October 2020 , The ISME Journal)

   Abstract Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13 C and 2 H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO 2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis , and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO 2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and thatmore »acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.« less
4. Metabolomic Responses of Green Alga Chlamydomonas reinhardtii Exposed to Sublethal Concentrations of Inorganic and Methylmercury.

   https://doi.org/10.1021/ acs.est.0c08416  

   Slaveykova, Vera I. ; Majumdar, Sanghamitra ; Regier, Nicole ; Li, Weiwei ; Keller, Arturo A. ( February 2021 , Environmental science and technology)

   Metabolomics characterizes low-molecular-weight molecules involved in different biochemical reactions and provides an integrated assessment of the physiological state of an organism. By using liquid chromatography–mass spectrometry targeted metabolomics, we examined the response of green alga Chlamydomonas reinhardtii to sublethal concentrations of inorganic mercury (IHg) and monomethylmercury (MeHg). We quantified the changes in the levels of 93 metabolites preselected based on the disturbed metabolic pathways obtained in a previous transcriptomics study. Metabolites are downstream products of the gene transcription; hence, metabolite quantification provided information about the biochemical status of the algal cells exposed to Hg compounds. The results showed that the alga adjusts its metabolism during 2 h exposure to 5 × 10–9 and 5 × 10–8 mol L–1 IHg and MeHg by increasing the level of various metabolites involved in amino acid and nucleotide metabolism, photorespiration, and tricarboxylic acid (TCA) cycle, as well as the metabolism of fatty acids, carbohydrates, and antioxidants. Most of the metabolic perturbations in the alga were common for IHg and MeHg treatments. However, the exposure to IHg resulted in more pronounced perturbations in the fatty acid and TCA metabolism as compared with the exposure to MeHg. The observed metabolic perturbations were generally consistent with ourmore »previously published transcriptomics results for C. reinhardtii exposed to the comparable level of IHg and MeHg. The results highlight the potential of metabolomics for toxicity evaluation, especially to detect effects at an early stage of exposure prior to their physiological appearance.« less
5. Functional differentiation determines the molecular basis of the symbiotic lifestyle of Ca. Nanohaloarchaeota

   https://doi.org/10.1186/s40168-022-01376-y  

   Xie, Yuan-Guo ; Luo, Zhen-Hao ; Fang, Bao-Zhu ; Jiao, Jian-Yu ; Xie, Qi-Jun ; Cao, Xing-Ru ; Qu, Yan-Ni ; Qi, Yan-Lin ; Rao, Yang-Zhi ; Li, Yu-Xian ; et al ( October 2022 , Microbiome)

   CandidatusNanohaloarchaeota, an archaeal phylum within the DPANN superphylum, is characterized by limited metabolic capabilities and limited phylogenetic diversity and until recently has been considered to exclusively inhabit hypersaline environments due to an obligate association withHalobacteria. Aside from hypersaline environments,Ca.Nanohaloarchaeota can also have been discovered from deep-subsurface marine sediments.

   Three metagenome-assembled genomes (MAGs) representing a new order within theCa.Nanohaloarchaeota were reconstructed from a stratified salt crust and proposed to represent a novel order,Nucleotidisoterales. Genomic features reveal them to be anaerobes capable of catabolizing nucleotides by coupling nucleotide salvage pathways with lower glycolysis to yield free energy. Comparative genomics demonstrated that these and otherCa.Nanohaloarchaeota inhabiting saline habitats use a “salt-in” strategy to maintain osmotic pressure based on the high proportion of acidic amino acids. In contrast, previously describedCa.Nanohaloarchaeota MAGs from geothermal environments were enriched with basic amino acids to counter heat stress. Evolutionary history reconstruction revealed that functional differentiation of energy conservation strategies drove diversification withinCa.Nanohaloarchaeota, further leading to shifts in the catabolic strategy from nucleotide degradation within deeper lineages to polysaccharide degradation within shallow lineages.

   This study provides deeper insight into the ecological functions and evolution of the expanded phylumCa.Nanohaloarchaeota and further advances our understanding on the functional and geneticmore »associations between potential symbionts and hosts.

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