Metabolic control of the Escherichia coli universal stress protein response through fructose-6-phosphate.

The universal stress protein (Usp) superfamily encompasses a conserved group of proteins involved in stress resistance, adaptation to energy deficiency, cell motility and adhesion, and is found in all kingdoms of life. The paradigm usp gene, uspA, of Escherichia coli is transcriptionally activated by a large variety of stresses, and the alarmone ppGpp is required for this activation. Here, we show that the uspA gene is also regulated by an intermediate of the glycolytic/gluconeogenic pathways. Specifically, mutations and conditions resulting in fructose-6-phosphate (F-6-P) accumulation elicit superinduction of uspA upon carbon starvation, whereas genetic manipulations reducing the pool size of F-6-P have the opposite effect. This metabolic control of uspA does not act via ppGpp. Other, but not all, usp genes of the usp superfamily are similarly affected by alterations in F-6-P levels. We suggest that alterations in the pool size of phosphorylated sugars of the upper glycolytic pathway may ensure accumulation of required survival proteins preceding the complete depletion of the external carbon source. Indeed, we show that uspA is, in fact, induced before the carbon source is depleted from the medium.

Distinct intracellular localization of Gpd1p and Gpd2p, the two yeast isoforms of NAD+-dependent glycerol-3-phosphate dehydrogenase, explains their different contributions to redox-driven glycerol production.

During anaerobiosis Saccharomyces cerevisiae strongly increases glycerol production to provide for non-respiratory oxidation of NADH to NAD(+). We here report that respiratory-deficient cells become strictly dependent on the Gpd2p isoform of the NAD(+)-linked glycerol-3-phosphate dehydrogenase (Gpd). The growth inhibition of respiratory incompetent cox18Delta cells lacking GPD2 is reversed by the addition of acetoin, an alternative sink for NADH oxidation. Growth is also restored by addition of lysine or glutamic acid/glutamine, the synthesis of which involves production of mitochondrial NADH. Lysine produced a stronger growth stimulating effect than glutamic acid consistent with an upregulated expression of the IDP3 gene for peroxisomal synthesis of the glutamate precursor alpha-ketoglutarate. Gpd2p is known to be a cytosolic protein but possesses a classical mitochondrial presequence, which we show is sufficient for mitochondrial targeting. A partial mitochondrial localization of Gpd2p will provide for establishment of intramitochondrial redox balance under non-respiratory conditions. Gpd1p, the other Gpd isoform, is partly cytosolic and partly peroxisomal and becomes more strictly peroxisomal in respiratory-deficient mutants. The different cellular distribution of Gpd1p and Gpd2p thus appears to be the main reason Gpd1p cannot substitute for Gpd2p in cox18Deltagpd2Delta cells, despite similar kinetic characteristics of the two iso-enzymes.

NADH-reductive stress in Saccharomyces cerevisiae induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (TDH1).

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2Delta strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2Delta strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2Delta strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.