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The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

ABSTRACT: What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

SUBMITTER: Sudarsan S

PROVIDER: S-EPMC4136102 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

Sudarsan Suresh S Dethlefsen Sarah S Blank Lars M LM Siemann-Herzberg Martin M Schmid Andreas A

Applied and environmental microbiology 20140620 17

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative car ...[more]

PMID: 24951791

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Project description:Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. This study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

Dataset Information

The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

Publications

The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

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