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Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli.

Goodarzi H, Bennett BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S - Mol. Syst. Biol. (2010)

Bottom Line: However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little.A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance.Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

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Ethanol degradation as a mechanism for tolerance. (A) The fitness profile of the genes active in the TCA cycle along with propionate catabolism genes (prp genes) and the negative regulators of alcohol dehydrogenase (cafA and fruR). (B) fnr/arcA and cafA deletions, both separately and in combination, significantly boost the ethanol tolerance capacity of E. coli. These experiments were performed in triplicate. Error bars mark the minimum and maximum values.
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f5: Ethanol degradation as a mechanism for tolerance. (A) The fitness profile of the genes active in the TCA cycle along with propionate catabolism genes (prp genes) and the negative regulators of alcohol dehydrogenase (cafA and fruR). (B) fnr/arcA and cafA deletions, both separately and in combination, significantly boost the ethanol tolerance capacity of E. coli. These experiments were performed in triplicate. Error bars mark the minimum and maximum values.

Mentions: In addition to osmoregulatory transcription factors, we also identified other regulatory proteins with significant contributions to ethanol tolerance. The key regulators we identified include FNR/ArcA, PrpR (Figure 2), and CafA (Figure 5A). FNR and ArcA, controllers of the aerobic to anaerobic switch (Green and Paget, 2004), largely regulate the central carbon metabolism enzymes. On the other hand, cafA codes for ribonuclease G, which is involved in rRNA processing (Umitsuki et al, 2001). We asked whether the contributions from these loci are additive by combining deletions in fnr, arcA, and cafA. These deletions, which individually increase ethanol tolerance, result in a large cumulative effect (Figure 5B). Prior studies had shown a decrease in the fnr transcript level in the ethanol-tolerant strain LY01; however, this was hypothesized to contribute through increased osmoprotection (Gonzalez et al, 2003). Our observations in MG1655, on the other hand, suggest that the activity of central metabolic enzymes, as part of the FNR/ArcA regulon, is the key contributor to ethanol tolerance (Figure 5A).


Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli.

Goodarzi H, Bennett BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S - Mol. Syst. Biol. (2010)

Ethanol degradation as a mechanism for tolerance. (A) The fitness profile of the genes active in the TCA cycle along with propionate catabolism genes (prp genes) and the negative regulators of alcohol dehydrogenase (cafA and fruR). (B) fnr/arcA and cafA deletions, both separately and in combination, significantly boost the ethanol tolerance capacity of E. coli. These experiments were performed in triplicate. Error bars mark the minimum and maximum values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913397&req=5

f5: Ethanol degradation as a mechanism for tolerance. (A) The fitness profile of the genes active in the TCA cycle along with propionate catabolism genes (prp genes) and the negative regulators of alcohol dehydrogenase (cafA and fruR). (B) fnr/arcA and cafA deletions, both separately and in combination, significantly boost the ethanol tolerance capacity of E. coli. These experiments were performed in triplicate. Error bars mark the minimum and maximum values.
Mentions: In addition to osmoregulatory transcription factors, we also identified other regulatory proteins with significant contributions to ethanol tolerance. The key regulators we identified include FNR/ArcA, PrpR (Figure 2), and CafA (Figure 5A). FNR and ArcA, controllers of the aerobic to anaerobic switch (Green and Paget, 2004), largely regulate the central carbon metabolism enzymes. On the other hand, cafA codes for ribonuclease G, which is involved in rRNA processing (Umitsuki et al, 2001). We asked whether the contributions from these loci are additive by combining deletions in fnr, arcA, and cafA. These deletions, which individually increase ethanol tolerance, result in a large cumulative effect (Figure 5B). Prior studies had shown a decrease in the fnr transcript level in the ethanol-tolerant strain LY01; however, this was hypothesized to contribute through increased osmoprotection (Gonzalez et al, 2003). Our observations in MG1655, on the other hand, suggest that the activity of central metabolic enzymes, as part of the FNR/ArcA regulon, is the key contributor to ethanol tolerance (Figure 5A).

Bottom Line: However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little.A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance.Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

Show MeSH
Related in: MedlinePlus