13C-flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis.

ABSTRACT: In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ?20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

SUBMITTER: Ruhl M 

PROVIDER: S-EPMC3431622 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature