Large-scale 13C-flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Autor: Uwe Sauer, Bart R. B. Haverkorn van Rijsewijk, Sophie Nallet, Roelco J. Kleijn, Annik Nanchen
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Zdroj: Molecular Systems Biology
Molecular Systems Biology, 7 (1)
DOI: 10.3929/ethz-b-000162898
Popis: The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways.
In contrast to the canonical respiro-fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the PEP-glyoxylate cycle. Of 91 transcription factors, 2/3 affect absolute fluxes, but only one controls the distribution of fluxes on galactose and nine on glucose. Transcriptional control of hexose flux distributions is confined to the acetyl-CoA branch point. The PEP-glyoxylate cycle is controlled by cAMP-Crp in a hexose uptake rate-dependent manner.
Focusing on central carbon metabolism of Escherichia coli, we aim here to systematically identify transcriptional regulators that control the distribution of metabolic fluxes during aerobic growth on hexoses. To assess the condition dependence of transcriptional control of flux, we selected glucose and galactose as two substrates that are highly similar, yet lead to distinct growth rates (Soupene et al, 2003), overall metabolic rates (De Anda et al, 2006; Samir El et al, 2009) and levels of catabolite repression (Hogema et al, 1998; Bettenbrock et al, 2007). Experimentally determined fluxes (Fischer and Sauer, 2003a) during growth on glucose and galactose reveal two distinct metabolic states. On glucose, high metabolic rates lead to high overflow metabolism and respiratory fluxes through the TCA cycle. On galactose, in contrast, metabolism was much slower without overflow metabolism and respiratory fluxes exclusively through the PEP-glyoxylate cycle (Fischer and Sauer, 2003b). To determine which transcriptional events controlled these two distinct metabolic states, we determined intracellular fluxes in 91 transcription factor mutants. These genetic perturbations primarily affected absolute fluxes but not the distribution of fluxes. The distribution of flux between glycolysis and pentose–phosphate pathway in upper metabolism, e.g., remained constant in all mutants under all conditions. Transcriptional control of the flux distribution was exclusively seen at the acetyl-CoA branch point. On glucose, nine transcription factors controlled the distribution of fluxes at this branch point, five of which (ArcA, IHFA, IHFB, PdhR, Fur) did so presumably directly through their known targets in TCA cycle and/or respiration. Without known targets in the relevant pathways, the remaining four transcription factors (GlpR, QseB, HdfR, GlcC) may act either indirectly or directly through unknown targets. On galactose, transcriptional control focused exclusively on the PEP-glyoxylate cycle. While deletion of six transcription factors (Cra, Crp, IHFA, IHFB, Mlc, NagC) abolished or reduced the PEP-glyoxylate cycle flux, we demonstrate by substrate-limited chemostat experiments, derepression of galactose uptake and show by metabolomics that five of these transcription factors act indirectly through increased cAMP concentrations that allosterically activate Crp, the only direct transcription factors that controls the PEP-glyoxylate cycle (Nanchen et al, 2008). Overall, our absolute flux data demonstrate that control of flux splitting during growth on hexoses was confined to the acetyl-CoA branch point in E. coli. Of the 36 transcription factors known to target genes in pathways that diverge from the acetyl-CoA branch point, only one transcription factor on galactose and five plus potentially four others on glucose showed altered flux splitting. The primary focus of steady state transcriptional control on the acetyl-CoA branch point, and thus the metabolic decision between the energetically efficient respiration and the less efficient but more rapid fermentation, was recently also demonstrated with only relative flux data for Saccharomyces cerevisiae (Fendt et al, 2010). In contrast to glucose-grown Bacillus subtilis (Fischer et al, 2005), for batch glucose-grown E. coli, none of the investigated transcription factor mutants exhibited improved biomass productivity. However, the mutants Cra, IHF A, IHF B and NagC with increased uptake rates grew much faster at almost unaltered biomass yields. As the removal of the glucose PTS-based repression with a Crr mutant also resulted in increased galactose uptake, we provide evidence that E. coli actively represses its galactose uptake at the expense of otherwise possible rapid growth.
Despite our increasing topological knowledge on regulation networks in model bacteria, it is largely unknown which of the many co-occurring regulatory events actually control metabolic function and the distribution of intracellular fluxes. Here, we unravel condition-dependent transcriptional control of Escherichia coli metabolism by large-scale 13C-flux analysis in 91 transcriptional regulator mutants on glucose and galactose. In contrast to the canonical respiro-fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the phosphoenol-pyruvate (PEP)-glyoxylate cycle. While 2/3 of the regulators directly or indirectly affected absolute flux rates, the partitioning between different pathways remained largely stable with transcriptional control focusing primarily on the acetyl-CoA branch point. Flux distribution control was achieved by nine transcription factors on glucose, including ArcA, Fur, PdhR, IHF A and IHF B, but was exclusively mediated by the cAMP-dependent Crp regulation of the PEP-glyoxylate cycle flux on galactose. Five further transcription factors affected this flux only indirectly through cAMP and Crp by increasing the galactose uptake rate. Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth.
Databáze: OpenAIRE
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