Limits...
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.

Show MeSH

Related in: MedlinePlus

Metabolite concentration measurements and stable-isotope labeling of ethanol-tolerant strains. (A) The steady-state level of UDP-glucose and UDP-N-acetyl-glucoseamine in ethanol-tolerant strains (HG227 and HG228) compared with wild-type MG1655. The samples were assayed in quadruplicate, and the error bars mark the s.d. (B) A significantly higher concentration of 2,3-dihydroxybenzoate was observed in HG228 compared with wild type. Also, the genes in the ent pathway have negative fitness scores in the fitness profiling of transposon insertion libraries. (C) Shown are the fraction of citrate/isocitrate or succinate metabolites that are labeled with carbon from the 13C-ethanol that was added at time 0 in both HG228 and wild-type strain MG1655. The samples were measured in triplicate with the error bars marking the s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2913397&req=5

f7: Metabolite concentration measurements and stable-isotope labeling of ethanol-tolerant strains. (A) The steady-state level of UDP-glucose and UDP-N-acetyl-glucoseamine in ethanol-tolerant strains (HG227 and HG228) compared with wild-type MG1655. The samples were assayed in quadruplicate, and the error bars mark the s.d. (B) A significantly higher concentration of 2,3-dihydroxybenzoate was observed in HG228 compared with wild type. Also, the genes in the ent pathway have negative fitness scores in the fitness profiling of transposon insertion libraries. (C) Shown are the fraction of citrate/isocitrate or succinate metabolites that are labeled with carbon from the 13C-ethanol that was added at time 0 in both HG228 and wild-type strain MG1655. The samples were measured in triplicate with the error bars marking the s.d.

Mentions: In addition to metabolite measurements, we also performed gene-expression profiling to compare transcript abundances in HG228 and WT. The expression values serve as additional information for analyzing the metabolite pool sizes. Remarkably, and consistent with our prior discoveries, HG228 shows a significant downregulation in acid stress response genes accompanied by upregulations in peptidoglycan biosynthesis, glycine cleavage system, ArcA regulon, and heat-shock stress response pathways (see Supplementary Figure S6). As mentioned in the earlier sections, the cell-wall biogenesis pathway is beneficial for ethanol tolerance. Both ethanol-tolerant strains HG227 and HG228 show significant reductions in the steady-state concentrations of UDP-glucose and UDP-N-acetyl-glucosamine, whereas HG227 also shows a significant reduction in UDP-glucuronate, indicating an increase in their consumption by peptidoglycan and colanic acid biosynthesis (Figure 7A). The higher expression of genes functioning in peptidoglycan biosynthesis pathway in HG228 further underlines an increase in cell-wall biogenesis.


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)

Metabolite concentration measurements and stable-isotope labeling of ethanol-tolerant strains. (A) The steady-state level of UDP-glucose and UDP-N-acetyl-glucoseamine in ethanol-tolerant strains (HG227 and HG228) compared with wild-type MG1655. The samples were assayed in quadruplicate, and the error bars mark the s.d. (B) A significantly higher concentration of 2,3-dihydroxybenzoate was observed in HG228 compared with wild type. Also, the genes in the ent pathway have negative fitness scores in the fitness profiling of transposon insertion libraries. (C) Shown are the fraction of citrate/isocitrate or succinate metabolites that are labeled with carbon from the 13C-ethanol that was added at time 0 in both HG228 and wild-type strain MG1655. The samples were measured in triplicate with the error bars marking the s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Metabolite concentration measurements and stable-isotope labeling of ethanol-tolerant strains. (A) The steady-state level of UDP-glucose and UDP-N-acetyl-glucoseamine in ethanol-tolerant strains (HG227 and HG228) compared with wild-type MG1655. The samples were assayed in quadruplicate, and the error bars mark the s.d. (B) A significantly higher concentration of 2,3-dihydroxybenzoate was observed in HG228 compared with wild type. Also, the genes in the ent pathway have negative fitness scores in the fitness profiling of transposon insertion libraries. (C) Shown are the fraction of citrate/isocitrate or succinate metabolites that are labeled with carbon from the 13C-ethanol that was added at time 0 in both HG228 and wild-type strain MG1655. The samples were measured in triplicate with the error bars marking the s.d.
Mentions: In addition to metabolite measurements, we also performed gene-expression profiling to compare transcript abundances in HG228 and WT. The expression values serve as additional information for analyzing the metabolite pool sizes. Remarkably, and consistent with our prior discoveries, HG228 shows a significant downregulation in acid stress response genes accompanied by upregulations in peptidoglycan biosynthesis, glycine cleavage system, ArcA regulon, and heat-shock stress response pathways (see Supplementary Figure S6). As mentioned in the earlier sections, the cell-wall biogenesis pathway is beneficial for ethanol tolerance. Both ethanol-tolerant strains HG227 and HG228 show significant reductions in the steady-state concentrations of UDP-glucose and UDP-N-acetyl-glucosamine, whereas HG227 also shows a significant reduction in UDP-glucuronate, indicating an increase in their consumption by peptidoglycan and colanic acid biosynthesis (Figure 7A). The higher expression of genes functioning in peptidoglycan biosynthesis pathway in HG228 further underlines an increase in cell-wall biogenesis.

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