Photoautotrophic Growth Rate Enhancement of Synechocystis sp. PCC6803 by Heterologous Production of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Chlorobaculum tepidum.

2-Oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR) is a carbon-fixing enzyme in the reductive TCA cycle that reversibly carboxylates succinyl-CoA to yield 2-oxoglutarate. CtOGOR is a heterotetramer of two large (α = 68 kDa) and two small (ß = 38 kDa) subunits. The αß protomer harbors one thiamine pyrophosphate and two 4Fe-4S clusters. Nonetheless, the enzyme has a considerable oxygen tolerance with a half-life of 143 min at 215 µM dissolved oxygen. Kinetic analyses of the purified recombinant CtOGOR revealed a lower Km for succinyl-CoA than for 2-oxoglutarate. Cellular levels of 2-oxoglutarate and glutamate­a product of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase­increased more than twofold in the exponential phase compared with the control strain, leading to an approximately >30% increase in the photoautotrophic growth rate. Thus, CtOGOR was successfully produced in Synechocystis, thereby boosting carboxylation, resulting in enhanced photoautotrophic growth.

Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion.

Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.

New viral biogeochemical roles revealed through metagenomic analysis of Lake Baikal.

BACKGROUND: Lake Baikal is the largest body of liquid freshwater on Earth. Previous studies have described the microbial composition of this habitat, but the viral communities from this ecosystem have not been characterized in detail. RESULTS: Here, we describe the viral diversity of this habitat across depth and seasonal gradients. We discovered 19,475 bona fide viral sequences, which are derived from viruses predicted to infect abundant and ecologically important taxa that reside in Lake Baikal, such as Nitrospirota, Methylophilaceae, and Crenarchaeota. Diversity analysis revealed significant changes in viral community composition between epipelagic and bathypelagic zones. Analysis of the gene content of individual viral populations allowed us to describe one of the first bacteriophages that infect Nitrospirota, and their extensive repertoire of auxiliary metabolic genes that might enhance carbon fixation through the reductive TCA cycle. We also described bacteriophages of methylotrophic bacteria with the potential to enhance methanol oxidation and the S-adenosyl-L-methionine cycle. CONCLUSIONS: These findings unraveled new ways by which viruses influence the carbon cycle in freshwater ecosystems, namely, by using auxiliary metabolic genes that act upon metabolisms of dark carbon fixation and methylotrophy. Therefore, our results shed light on the processes through which viruses can impact biogeochemical cycles of major ecological relevance. Video Abstract.