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Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions.

Moreau PL, Loiseau L - Microb. Cell Fact. (2016)

Bottom Line: We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures.Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption.Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Bactérienne, UMR 7283, Aix-Marseille Université, Marseille, France. moreau@imm.cnrs.fr.

ABSTRACT

Background: During prolonged incubation of Escherichia coli K-12 in batch culture under aerobic, phosphate (Pi) starvation conditions, excess glucose is converted into acetic acid, which may trigger cell death. Following serial cultures, we isolated five evolved strains in two populations that survived prolonged incubation.

Methods: We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures.

Results: Evolved strains used two main strategies: (1) the constitutive expression of the Trk- and Kdp-dependent K(+) transport systems, and (2) the inactivation of the ArcA global regulator. Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption. Evolved strains acquired a few additional genetic changes besides the trkH, kdpD and arcA mutations, which might increase the scavenging of organophosphates (phnE (+), lapB, and rseP) and the resistance to oxidative (rsxC) and acetic acid stresses (e14(-)/icd (+)).

Conclusions: Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability. The spread of these mutant strains might give the ancestral strain time to accumulate up to five genetic changes (phnE (+) arcA rsxC crfC e14(-)/icd (+)), which allowed growth on glucose and organophosphates, and provided a long-term survival. The latter strain, which expressed several mechanisms of protection against endogenous and exogenous stresses, might provide a platform for producing toxic recombinant proteins and chemicals during prolonged incubation under aerobic, Pi starvation conditions.

No MeSH data available.


Related in: MedlinePlus

Schematic illustration of the relationship between pyruvate metabolism and the aerobic respiratory chain. AceCoA acetyl CoA, BCAA branched-chain amino acids, CIT citrate, CS citrate synthase (GltA), FAD flavin adenine dinucleotide, FUM fumarate, GABA γ-aminobutyrate, Glc glucose, Glg glycogen, GLX glyoxylate, GLU glutamate, G6P glucose 6-phosphate, ICT isocitrate, IDH isocitrate dehydrogenase (Icd), IM inner membrane, KG α-ketoglutarate, KGDH α-ketoglutarate dehydrogenase, MAL malate, MDH malate dehydrogenase, NDH NADH dehydrogenase, OA oxaloacetate, PDH pyruvate dehydrogenase, pHi internal pH, PEP phosphoenolpyruvate, PYR pyruvate, Q ubiquinone, SDH succinate dehydrogenase, SUC succinate, SucCoA succinyl CoA; TCA, tricarboxylic acid
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Fig1: Schematic illustration of the relationship between pyruvate metabolism and the aerobic respiratory chain. AceCoA acetyl CoA, BCAA branched-chain amino acids, CIT citrate, CS citrate synthase (GltA), FAD flavin adenine dinucleotide, FUM fumarate, GABA γ-aminobutyrate, Glc glucose, Glg glycogen, GLX glyoxylate, GLU glutamate, G6P glucose 6-phosphate, ICT isocitrate, IDH isocitrate dehydrogenase (Icd), IM inner membrane, KG α-ketoglutarate, KGDH α-ketoglutarate dehydrogenase, MAL malate, MDH malate dehydrogenase, NDH NADH dehydrogenase, OA oxaloacetate, PDH pyruvate dehydrogenase, pHi internal pH, PEP phosphoenolpyruvate, PYR pyruvate, Q ubiquinone, SDH succinate dehydrogenase, SUC succinate, SucCoA succinyl CoA; TCA, tricarboxylic acid

Mentions: At the approach of the stationary phase, the cells steadily accumulate the RpoS (σs) sigma factor [6, 7]. The σs-RNA polymerase holoenzyme poorly transcribes “growth genes” (e.g. genes encoding the succinate dehydrogenase of the tricarboxylic acid cycle) and preferentially transcribes “defense genes” required for the protection of non-growing cells against endogenous stresses such as oxidative stress (e.g. pdhR, poxB, and sodC) and acetic acid stress (e.g. gadB) [4, 8–10]. For instance, cells incubated under Pi starvation conditions continue to metabolize glucose at a reduced rate, but eventually redirect the metabolic flux from the pyruvate dehydrogenase towards the RpoS-dependent pyruvate oxidase, PoxB, which directly converts pyruvate into acetic acid. PoxB (pyruvate:Q reductase), in contrast to the pyruvate dehydrogenase, does not use NAD+ as a cofactor, which prevents the adventitious production of O2.− and H2O2 by NADH dehydrogenases in the aerobic respiratory chain [4]. Whereas the activity of PoxB protects Pi-starved cells against oxidative stress at the entry into stationary phase, this activity can eventually cause the accumulation of high levels of acetic acid, which decrease the internal pH (pHi), stop metabolism and trigger cell death [4, 10]. Death of Pi-starved cells can be alleviated by the addition of glutamate into the medium, which allows the RpoS-dependent GadB acid resistance system to neutralize acetic acid [5, 10] (Fig. 1).Fig. 1


Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions.

Moreau PL, Loiseau L - Microb. Cell Fact. (2016)

Schematic illustration of the relationship between pyruvate metabolism and the aerobic respiratory chain. AceCoA acetyl CoA, BCAA branched-chain amino acids, CIT citrate, CS citrate synthase (GltA), FAD flavin adenine dinucleotide, FUM fumarate, GABA γ-aminobutyrate, Glc glucose, Glg glycogen, GLX glyoxylate, GLU glutamate, G6P glucose 6-phosphate, ICT isocitrate, IDH isocitrate dehydrogenase (Icd), IM inner membrane, KG α-ketoglutarate, KGDH α-ketoglutarate dehydrogenase, MAL malate, MDH malate dehydrogenase, NDH NADH dehydrogenase, OA oxaloacetate, PDH pyruvate dehydrogenase, pHi internal pH, PEP phosphoenolpyruvate, PYR pyruvate, Q ubiquinone, SDH succinate dehydrogenase, SUC succinate, SucCoA succinyl CoA; TCA, tricarboxylic acid
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4759930&req=5

Fig1: Schematic illustration of the relationship between pyruvate metabolism and the aerobic respiratory chain. AceCoA acetyl CoA, BCAA branched-chain amino acids, CIT citrate, CS citrate synthase (GltA), FAD flavin adenine dinucleotide, FUM fumarate, GABA γ-aminobutyrate, Glc glucose, Glg glycogen, GLX glyoxylate, GLU glutamate, G6P glucose 6-phosphate, ICT isocitrate, IDH isocitrate dehydrogenase (Icd), IM inner membrane, KG α-ketoglutarate, KGDH α-ketoglutarate dehydrogenase, MAL malate, MDH malate dehydrogenase, NDH NADH dehydrogenase, OA oxaloacetate, PDH pyruvate dehydrogenase, pHi internal pH, PEP phosphoenolpyruvate, PYR pyruvate, Q ubiquinone, SDH succinate dehydrogenase, SUC succinate, SucCoA succinyl CoA; TCA, tricarboxylic acid
Mentions: At the approach of the stationary phase, the cells steadily accumulate the RpoS (σs) sigma factor [6, 7]. The σs-RNA polymerase holoenzyme poorly transcribes “growth genes” (e.g. genes encoding the succinate dehydrogenase of the tricarboxylic acid cycle) and preferentially transcribes “defense genes” required for the protection of non-growing cells against endogenous stresses such as oxidative stress (e.g. pdhR, poxB, and sodC) and acetic acid stress (e.g. gadB) [4, 8–10]. For instance, cells incubated under Pi starvation conditions continue to metabolize glucose at a reduced rate, but eventually redirect the metabolic flux from the pyruvate dehydrogenase towards the RpoS-dependent pyruvate oxidase, PoxB, which directly converts pyruvate into acetic acid. PoxB (pyruvate:Q reductase), in contrast to the pyruvate dehydrogenase, does not use NAD+ as a cofactor, which prevents the adventitious production of O2.− and H2O2 by NADH dehydrogenases in the aerobic respiratory chain [4]. Whereas the activity of PoxB protects Pi-starved cells against oxidative stress at the entry into stationary phase, this activity can eventually cause the accumulation of high levels of acetic acid, which decrease the internal pH (pHi), stop metabolism and trigger cell death [4, 10]. Death of Pi-starved cells can be alleviated by the addition of glutamate into the medium, which allows the RpoS-dependent GadB acid resistance system to neutralize acetic acid [5, 10] (Fig. 1).Fig. 1

Bottom Line: We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures.Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption.Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Bactérienne, UMR 7283, Aix-Marseille Université, Marseille, France. moreau@imm.cnrs.fr.

ABSTRACT

Background: During prolonged incubation of Escherichia coli K-12 in batch culture under aerobic, phosphate (Pi) starvation conditions, excess glucose is converted into acetic acid, which may trigger cell death. Following serial cultures, we isolated five evolved strains in two populations that survived prolonged incubation.

Methods: We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures.

Results: Evolved strains used two main strategies: (1) the constitutive expression of the Trk- and Kdp-dependent K(+) transport systems, and (2) the inactivation of the ArcA global regulator. Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption. Evolved strains acquired a few additional genetic changes besides the trkH, kdpD and arcA mutations, which might increase the scavenging of organophosphates (phnE (+), lapB, and rseP) and the resistance to oxidative (rsxC) and acetic acid stresses (e14(-)/icd (+)).

Conclusions: Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability. The spread of these mutant strains might give the ancestral strain time to accumulate up to five genetic changes (phnE (+) arcA rsxC crfC e14(-)/icd (+)), which allowed growth on glucose and organophosphates, and provided a long-term survival. The latter strain, which expressed several mechanisms of protection against endogenous and exogenous stresses, might provide a platform for producing toxic recombinant proteins and chemicals during prolonged incubation under aerobic, Pi starvation conditions.

No MeSH data available.


Related in: MedlinePlus