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1. Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

   https://doi.org/10.7554/eLife.94903.2  

   Shiraliyev, Rauf; Orman, Mehmet A (June 2024, eLife)

   Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells. 

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

   https://doi.org/10.1073/pnas.2016380117  

   Mendonca, Caroll M.; Yoshitake, Sho; Wei, Hua; Werner, Anne; Sasnow, Samantha S.; Thannhauser, Theodore W.; Aristilde, Ludmilla (December 2020, Proceedings of the National Academy of Sciences)

   null (Ed.)

   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. 

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3. A thermodynamic bottleneck in the TCA cycle contributes to acetate overflow in Staphylococcus aureus

   https://doi.org/10.1128/msphere.00883-24  

   Shahreen, Nabia; Ahn, Jongsam; Alsiyabi, Adil; Chowdhury, Niaz Bahar; Shinde, Dhananjay; Chaudhari, Sujata S; Bayles, Kenneth W; Thomas, Vinai C; Saha, Rajib (January 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT During aerobic growth,S. aureusrelies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow inS. aureushave not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient forS. aureusto generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck providesS. aureusthe flexibility to redirect carbon toward maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offersS. aureusdistinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal–pathogenic lifestyle. 

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4. The impact of aminoglycoside exposure on soil and plant root-associated microbiota: a meta-analysis

   https://doi.org/10.1186/s13750-025-00365-6  

   Coates, Jessica_L; Lawson, Ashanti_J; Bostick, Kathleen; Ayalew, Mentewab (July 2025, Environmental Evidence)

   Abstract BackgroundExposure to aminoglycosides, a class of potent bactericidal antibiotics naturally produced by soil microorganisms and commonly used in agriculture, has the potential to cause shifts in the population dynamics of microorganisms that impact plant and soil health. In particular, aminoglycoside exposure could result in alterations of the soil and plant root-associated bacterial species diversity and richness due to their potent inhibitory action on microbial growth, the creation of selective conditions for the proliferation of antibiotic-resistant bacteria, or a reduction in the ability to suppress soil pathogens. Previous studies have attempted to understand the relationship between aminoglycoside exposure and the plant-associated microbiota with varying results. Thus, this systematic review aims to survey all relevant published data to answer the question, “What is the impact of aminoglycoside exposure on the soil and plant root-associated microbiota?” MethodsWe searched 5 academic databases and 1 specialist organization database for scientific journal publications written in any language. Articles were included based on the criteria described in Coates et al., 2022. Included studies were subject to critical appraisal using the CEE Critical Appraisal Tool Version 0.2 (Prototype) to evaluate their susceptibility to confounding factors, misclassification bias, selection bias, attrition bias, reporting bias and analysis bias. Studies deemed to be high risk based on critical appraisal results were excluded from further analysis. Descriptive data analysis was performed for studies considered low or unclear for risk of bias. Meta-analyses were conducted for antibiotic resistance and microbial diversity. Review findingsOut of 8370 screened records, 50 articles fulfilled the search criteria, and from these, 13 studies were included in meta-analysis. Most studies investigated the impact of aminoglycoside exposure on soil microbiota (93%) in a laboratory setting (62%), primarily from the United States (32%), China (24%), France, Switzerland and Germany (8%). A limited number of studies investigated the impact of aminoglycoside exposure on disease suppression, so it was excluded from meta-analysis. Therefore, our synthesis primarily details the impact of aminoglycoside exposure on the microbial diversity and antibiotic resistance of the soil microbiota. Overall, exposure to aminoglycosides did not result in a significant change in the microbial diversity. However, soil use, pH, and type of aminoglycoside used could be potential modifiers. Additionally, we observed an average 7% of the microbial population exhibiting resistance to aminoglycosides, with the relationship between the exposure concentration and the selection concentration emerging as a potential modifier. ConclusionsCurrent research is limited by gaps in understanding the relationship between aminoglycoside exposure, microbial community dynamics, and disease suppression, as well as by insufficient data on less-studied aminoglycosides and key confounding factors. Current research also suggests a potential relationship between antibiotic concentrations used for exposure and selection of resistant bacteria. These findings emphasize the need for informed antibiotic management policies and rigorous, targeted research to better understand the relationship between soil factors and antibiotic concentrations used on the impact of aminoglycosides on soil microbiota. 

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5. Overexpression of peroxisome proliferator-activated receptor co-activator-1⍺ (PGC-1⍺) in Chinese hamster ovary cells increases oxidative metabolism and IgG productivity

   https://doi.org/10.1016/j.ymben.2023.07.005  

   Sacco, Sarah A.; McAtee Pereira, Allison G.; Trenary, Irina; Smith, Kevin D.; Betenbaugh, Michael J.; Young, Jamey D. (September 2023, Metabolic Engineering)

   Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor 𝛾 co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools. 

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