&lt;i&gt;Escherichia coli arcA&lt;/i&gt; Mutants: Metabolic Profile Characterization of Microaerobic Cultures using Glycerol as a Carbon Source

Nikel, Pablo I.; Pettinari, M. Julia; Ramírez, M.Cecilia; Galvagno, Miguel A.; Méndez, Beatriz S.

doi:10.1159/000111992

Cited by 51 publications

(34 citation statements)

References 62 publications

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“…The oxidation state of NAD(H) has previously been reported to vary greatly in different genetic backgrounds (49)(50)(51)(52), such as in redox regulatory mutants (53)(54)(55), so the differences in NADH/NAD ϩ ratios between the strains carrying the cre variants support the hypothesis that CreC affects the intracellular redox state. Additionally, the results obtained in this work could reflect the fact that the content of NADP ϩ responds more strongly to variations in oxygen availability than to different genetic or metabolic backgrounds.…”

Section: Discussionmentioning

confidence: 88%

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Godoy

Nikel

Gomez

et al. 2016

Appl Environ Microbiol

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The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ⌬creC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ⌬creC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P) ؉ ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ⌬creC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ⌬creC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.T he survival of an organism depends, at least in part, on its ability to sense and respond to changes in the environment. In bacteria, global regulators control the transcription of genes in response to specific external stimuli and metabolic signals, finely tuning different aspects of their physiology to overcome environmental challenges. In Escherichia coli, seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate the expression of about one-half of all genes (1). This facultative aerobe is able to adapt its metabolism to different oxygen availability conditions through the concerted actions of a network of regulators, including the global regulators ArcAB and Fnr (2-4). These regulators affect many metabolic pathways, allowing the cells to reach an adequate redox balance under any given conditions. There is a very close association between carbon and electron flows, and even small differences in oxygen availability have been observed to elicit profound effects on the distribution of carbon fluxes (5).CreBC (for carbon source responsive) is a global sensing and regulation system affecting genes involved in a variety of functions, including enzymes of intermediary catabolism (6). Previous studies have shown that the creABCD operon is activated (i) during growth in minimal mediu...

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Section: Discussionmentioning

confidence: 88%

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Godoy

Nikel

Gomez

et al. 2016

Appl Environ Microbiol

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“…Oshima et al (50) also considered that the CreBC system does not exert an important regulation on gene expression under aerobic conditions, as judged from results of microarray transcriptomic experiments using E. coli mutants in all known two-component signal transduction systems. However, it is important to stress that both CreBC and ArcAB regulatory systems seem to exert an important influence on central metabolism in microaerobiosis (3,8,48,59), and this could be the reason for which no differences were observed under aerobic conditions of growth. Finally, and taking into account that both global regulatory systems exert an important influence on carbon and redox balances, manipulation of the genes for these regulators could provide a relevant tool for modulation of central metabolism and reducing power availability aimed at biotechnological purposes.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

et al. 2009

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Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of 13 C-labeling experiments in chemostat cultures of a wild-type strain, ⌬creB and ⌬arcA single mutants, and a ⌬creB ⌬arcA double mutant. Continuous cultures were conducted at D ‫؍‬ 0.1 h ؊1 under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the EmbdenMeyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its ⌬arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ⌬arcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.Transcriptional regulation comprises a complex network of global and specific regulators. Global regulators normally have pleiotropic effects on metabolism, since they control the transcription of several operons that belong to different functional groups. In Escherichia coli, seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate expression of about one-half of all genes (41). CreBC and ArcAB are two global sensing and regulation systems in E. coli which, along with some other regulatory systems, modulate the expression of genes involved in central metabolism and respiratory pathways allowing rapid adaptive responses to carbon source and oxygen availability in the culture environment.The cre (for carbon source responsive) locus comprises creABCD and was formerly known as the phoM locus (4). Whereas creA is a hypothetical open reading frame and creD, also known as cet, encodes an inner-membrane protein of unknown function, creB and creC encode a two-component system, i.e., a cytoplasmic regulator and a sensor kinase, respectively. After the discovery of CreBC, genes modulated by this system proved elusive. Based on sequence analysis and using bioinformatic tools Avison et al. (5) were able to define a cre tag sequence, to which CreB binds in vitro (8), in order to describe the...

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“…As a result, large amounts of reducing equivalents are available to be used in the synthesis of reduced compounds. Based on these facts, we studied the synthesis of two reduced bioproducts, poly(3-hydroxybutyrate) (18), which has the potential to be used as a biodegradable plastic, and ethanol (19), in a modified arcA genetic background. Two different mutations were analyzed: ⌬arcA and arcA2 (12), carried by strains CT1062 and CT1061, respectively, which are derivatives of E. coli K1060 [F Ϫ fadE62 lacI60 tyrT58 (AS) fabB5 mel-1] (20).…”

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confidence: 99%

“…arcA2 and ⌬arcA mutants were also observed to differ in other traits: arcA deletion mutants are very sensitive to redox dyes, such as toluidine blue (4), while strains bearing the arcA2 mutation produce medium-sized colonies on toluidine blue agar. Besides, arcA2 strains show a higher respiratory capacity (18,21) and show a higher NADH/NAD ϩ ratio when glycerol is used as a carbon substrate (19); as a consequence, enhanced yields of poly(3-hydroxybutyrate) and ethanol were observed. The different behaviors of these mutants were intriguing, and a deeper analysis was necessary in order to explain the molecular basis for the differences in phenotypes.…”

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The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an Escherichia coli arcA Mutant

et al. 2008

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Strains derived from HfrH carrying the arcA2 null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than arcA deletion mutants of a different lineage. The phenotype of the arcA2 mutants was due to the presence of a creC constitutive mutation introduced by P1 transduction.Escherichia coli and other facultative aerobes can adapt their metabolism according to O 2 availability by means of aerobic and anaerobic respiration and fermentation. ArcAB is a twocomponent signal transduction system that controls, at the transcriptional level, the choice of energy generation pathways according to the intracellular redox state (13,16,22). E. coli arc mutants are unregulated for aerobic respiration in microaerobic conditions, and so the enzymes of the tricarboxylic acid cycle are not repressed, and the pool of reducing equivalents (such as NADH or NADPH) is elevated (1, 24, 25). As a result, large amounts of reducing equivalents are available to be used in the synthesis of reduced compounds. Based on these facts, we studied the synthesis of two reduced bioproducts, poly(3-hydroxybutyrate) (18), which has the potential to be used as a biodegradable plastic, and ethanol (19), in a modified arcA genetic background. Two different mutations were analyzed: ⌬arcA and arcA2 (12), carried by strains CT1062 and CT1061, respectively, which are derivatives of E. coli K1060 [F Ϫ fadE62 lacI60 tyrT58 (AS) fabB5 mel-1] (20). Some ⌬arcA mutants are unable to grow in minimal medium (15), whereas arcA2 strains can grow in synthetic medium if it is supplemented with a low amount of casein amino acids to prime growth (9). arcA2 and ⌬arcA mutants were also observed to differ in other traits: arcA deletion mutants are very sensitive to redox dyes, such as toluidine blue (4), while strains bearing the arcA2 mutation produce medium-sized colonies on toluidine blue agar. Besides, arcA2 strains show a higher respiratory capacity (18,21) and show a higher NADH/NAD ϩ ratio when glycerol is used as a carbon substrate (19); as a consequence, enhanced yields of poly(3-hydroxybutyrate) and ethanol were observed. The different behaviors of these mutants were intriguing, and a deeper analysis was necessary in order to explain the molecular basis for the differences in phenotypes.arcA2 is a null mutation. Partial sequencing of the arcA2 allele had shown an IS10 insertion element in codon 170 (10). A more complete sequencing performed over an amplification fragment obtained from strain CT1061 revealed an IS10-L

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<i>Escherichia coli arcA</i> Mutants: Metabolic Profile Characterization of Microaerobic Cultures using Glycerol as a Carbon Source

Cited by 51 publications

References 62 publications

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an Escherichia coli arcA Mutant

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