Quantifying the Intracellular Metabolic Network that Establishes the Simultaneous Utilization of Sugars and Aromatic Substrates in Pseudomonas putida KT2440

The ability of Pseudomonas to use both aromatic compounds and sugars is being explored and exploited for emerging biotechnological applications, such as bioremediation as well as biofuel and chemical synthesis. The present study investigates the intracellular metabolism in the biotechnologically-important soil bacterium Pseudomonas putida KT2440 during feeding on a mixture containing the cellulosic hexose sugar (glucose) and an aromatic carboxylic acid (benzoate). Through a combination of 13C tracer experiments with metabolic flux analysis (MFA), I elucidated and quantified the discriminate metabolic routing of each substrate throughout central carbon metabolism. The results determined that glucose-derived carbon was primarily routed to the Entner-Doudoroff (ED) pathway, reverse Embden–Meyerhof–Parnas (EMP) pathway, and Pentose Phosphate (PP) pathway, while benzoate-derived carbon was routed almost exclusively to the tricarboxylic acid (TCA) cycle. I found that benzoate catabolism influenced the routing of glucose in the ED pathway, reverse EMP pathway, and PP pathway, despite the absence of benzoate-derived carbon in these pathways. In the TCA cycle, the simultaneous catabolism of benzoate and glucose led to the activation of the glyoxylate shunt. Accordingly, kinetic isotopic flux revealed a decreased flux through isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. In addition, feeding on the substrate mixture induced carbon overflow from the TCA cycle primarily through a significant involvement of malate dehydrogenase activity over the ED pathway in the biosynthesis of pyruvate. In sum, the data revealed that a preferential flux of substrate-derived carbons through different metabolic pathways was necessary to optimize biomass growth. These findings will contribute to an emerging framework for understanding soil carbon metabolism and advancing novel biotechnological applications in soil bacteria

Life Stage, Gender and Movement of Blue Crabs (Callinectis sapidus) in Lake Mattamuskeet and Connecting Canals

In their ranges on east and south coasts of the Americas as well as their established invasions in the Adriatic and Baltic, blue crabs, Callinectis sapidus, inhabit estuaries, sounds and coastal oceans and are commercially and ecologically important. How crabs move in response to physical variables is important to management. We monitored life stages at canal control structures, assessed gender ratios with recreational crabbing, learned from crabbers, and studied movements of tagged crabs in a canal connecting Lake Mattamuskeet to the Pamlico sound.  Juveniles enter the lake  through two of 4 canals connecting to the sounds.  Females migrate out through one canal. The lake standing population is about 70% male.  Movements of 240 crabs in August 2012 and 102 crabs in October 2014 were quantified using RFID tags with co-located meteorological and oceanographic devices. Non-spawning females and males are nomadic.  Crabs released in the canal move in response to changes in water depth and go with the flow, toward the Pamlico Sound (summer 76% and fall 78%). What crabbers describe as a fall migration  appears to be concentration of crabs in warmer deeper canals and then southern movement with flow generated by strong north winds. To be effective, management strategies like migratory corridors require understanding of crab movements

Glyphosate-Induced Specific and Widespread Perturbations in the Metabolome of Soil Pseudomonas Species

Previous studies have reported adverse effects of glyphosate on crop-beneficial soil bacterial species, including several soil Pseudomonas species. Of particular interest is the elucidation of the metabolic consequences of glyphosate toxicity in these species. Here we investigated the growth and metabolic responses of soil Pseudomonas species grown on succinate, a common root exudate, and glyphosate at different concentrations. We conducted our experiments with one agricultural soil isolate, P. fluorescens RA12, and three model species, P. putida KT2440, P. putida S12, and P. protegens Pf-5. Our results demonstrated both species- and strain-dependent growth responses to glyphosate. Following exposure to a range of glyphosate concentrations (up to 5 mM), the growth rate of both P. protegens Pf-5 and P. fluorescens RA12 remained unchanged whereas the two P. putida strains exhibited from 0 to 100% growth inhibition. We employed a 13C-assisted metabolomics approach using liquid chromatography-mass spectrometry to monitor disruptions in metabolic homeostasis and fluxes. Profiling of the whole-cell metabolome captured deviations in metabolite levels involved in the tricarboxylic acid cycle, ribonucleotide biosynthesis, and protein biosynthesis. Altered metabolite levels specifically in the biosynthetic pathway of aromatic amino acids (AAs), the target of toxicity for glyphosate in plants, implied the same toxicity target in the soil bacterium. Kinetic flux experiments with 13C-labeled succinate revealed that biosynthetic fluxes of the aromatic AAs were not inhibited in P. fluorescens Pf-5 in the presence of low and high glyphosate doses but these fluxes were inhibited by up to 60% in P. putida KT2440, even at sub-lethal glyphosate exposure. Notably, the greatest inhibition was found for the aromatic AA tryptophan, an important precursor to secondary metabolites. When the growth medium was supplemented with aromatic AAs, P. putida S12 exposed to a lethal dose of glyphosate completely recovered in terms of both growth rate and selected metabolite levels. Collectively, our findings led us to conclude that the glyphosate-induced specific disruption of de novo biosynthesis of aromatic AAs accompanied by widespread metabolic disruptions was responsible for dose-dependent adverse effects of glyphosate on sensitive soil Pseudomonas species

NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C

Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function, and neurodegeneration. The compositional and functional alterations in NPC lysosomes and nature of aberrant cholesterol-mTORC1 signaling contribution to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion, enhanced membrane damage, and defective mitophagy. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC