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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 overview of possible changes in ENZ1901 and ENZ1902. BCAA branched-chain amino acids, LPS lipopolysaccharide, PDH pyruvate dehydrogenase, PST phosphate transport, PYR pyruvate, R-O-P organophosphate, TCA tricarboxylic acid
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Fig14: Schematic overview of possible changes in ENZ1901 and ENZ1902. BCAA branched-chain amino acids, LPS lipopolysaccharide, PDH pyruvate dehydrogenase, PST phosphate transport, PYR pyruvate, R-O-P organophosphate, TCA tricarboxylic acid

Mentions: We found evidence that the lapB43 mutation, present in the evolved strain ENZ1901, might help growth on glucose and organophosphates (GPS phenotype), and eventually on acetate and Pi (Ace+ phenotype). LapB is an inner membrane protein that negatively controls the biosynthesis of the lipopolysaccharide [30, 31]. Because the lipopolysaccharide is negatively charged as a result of the presence of two phosphates in lipid A [54], we suggest that the lipopolysaccharide might hamper the binding to the outer membrane of negatively charged products such as Pi and organophosphates. Therefore, we speculate that lapB43 might be a gain-of-function mutation that would decrease the levels of the lipopolysaccharide, which might increase the binding and the diffusion of Pi and organophosphates required for the expression of the GPS phenotype (Fig. 14). The binding of P compounds to the outer membrane may be critical in rpoS+ cells in which the induction of the Pho regulon and the synthesis of P-compound scavengers are very transient [25]. In this light, the acquisition of the rpoS214- mutation in the phnE+lapB43 strain might favor the GPS phenotype in two different ways: by rerouting the metabolic flux towards the tricarboxylic acid cycle [1] and by extending the expression of the Pho regulon [25]. Unfortunately, the rpoS214 mutation reduced the long-term viability of the evolved strain ENZ1901 in mixed culture; this may reflect a defect in acetic acid resistance through the so-called RpoS-dependent amino acid-independent “acid resistance system 1” [55].Fig. 14


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

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

Schematic overview of possible changes in ENZ1901 and ENZ1902. BCAA branched-chain amino acids, LPS lipopolysaccharide, PDH pyruvate dehydrogenase, PST phosphate transport, PYR pyruvate, R-O-P organophosphate, TCA tricarboxylic acid
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig14: Schematic overview of possible changes in ENZ1901 and ENZ1902. BCAA branched-chain amino acids, LPS lipopolysaccharide, PDH pyruvate dehydrogenase, PST phosphate transport, PYR pyruvate, R-O-P organophosphate, TCA tricarboxylic acid
Mentions: We found evidence that the lapB43 mutation, present in the evolved strain ENZ1901, might help growth on glucose and organophosphates (GPS phenotype), and eventually on acetate and Pi (Ace+ phenotype). LapB is an inner membrane protein that negatively controls the biosynthesis of the lipopolysaccharide [30, 31]. Because the lipopolysaccharide is negatively charged as a result of the presence of two phosphates in lipid A [54], we suggest that the lipopolysaccharide might hamper the binding to the outer membrane of negatively charged products such as Pi and organophosphates. Therefore, we speculate that lapB43 might be a gain-of-function mutation that would decrease the levels of the lipopolysaccharide, which might increase the binding and the diffusion of Pi and organophosphates required for the expression of the GPS phenotype (Fig. 14). The binding of P compounds to the outer membrane may be critical in rpoS+ cells in which the induction of the Pho regulon and the synthesis of P-compound scavengers are very transient [25]. In this light, the acquisition of the rpoS214- mutation in the phnE+lapB43 strain might favor the GPS phenotype in two different ways: by rerouting the metabolic flux towards the tricarboxylic acid cycle [1] and by extending the expression of the Pho regulon [25]. Unfortunately, the rpoS214 mutation reduced the long-term viability of the evolved strain ENZ1901 in mixed culture; this may reflect a defect in acetic acid resistance through the so-called RpoS-dependent amino acid-independent “acid resistance system 1” [55].Fig. 14

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