The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

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A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02376-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 564-573 ◽

Cited By ~ 24

Author(s):

Nobutaka Nakashima ◽

Satoshi Ohno ◽

Katsunori Yoshikawa ◽

Hiroshi Shimizu ◽

Tomohiro Tamura

Keyword(s):

Escherichia Coli ◽

Carbon Metabolism ◽

Target Genes ◽

Central Carbon Metabolism ◽

Accurate Information ◽

Content Type ◽

E Coli ◽

Antisense Rnas ◽

Central Carbon ◽

Multiple Gene Silencing

ABSTRACTWe describe here the construction of a series of 71 vectors to silence central carbon metabolism genes inEscherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared toin silicopredictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that anyE. colistrain can be used and multiple gene silencing is easily possible in any combination.

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Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism

PLoS ONE ◽

10.1371/journal.pone.0176050 ◽

2017 ◽

Vol 12 (4) ◽

pp. e0176050 ◽

Cited By ~ 7

Author(s):

Joanna Tymecka-Mulik ◽

Lidia Boss ◽

Monika Maciąg-Dorszyńska ◽

João F. Matias Rodrigues ◽

Lidia Gaffke ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Carbon Metabolism ◽

Central Carbon Metabolism ◽

Central Carbon ◽

Replication Regulation ◽

Dnaa46 Mutation

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Multiple links connect central carbon metabolism to DNA replication initiation and elongation inBacillus subtilis

DNA Research ◽

10.1093/dnares/dsy031 ◽

2018 ◽

Vol 25 (6) ◽

pp. 641-653 ◽

Cited By ~ 4

Author(s):

Hamid Nouri ◽

Anne-Françoise Monnier ◽

Solveig Fossum-Raunehaug ◽

Monika Maciąg-Dorszyńska ◽

Armelle Cabin-Flaman ◽

...

Keyword(s):

Dna Replication ◽

Carbon Metabolism ◽

Central Carbon Metabolism ◽

Replication Initiation ◽

Dna Replication Initiation ◽

Central Carbon

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Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

Journal of Bacteriology ◽

10.1128/jb.181.21.6679-6688.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6679-6688 ◽

Cited By ~ 263

Author(s):

Uwe Sauer ◽

Daniel R. Lasko ◽

Jocelyne Fiaux ◽

Michel Hochuli ◽

Ralf Glaser ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Metabolism ◽

Metabolic Flux ◽

Flux Ratio ◽

Tca Cycle ◽

Central Carbon Metabolism ◽

E Coli ◽

Chemostat Cultures ◽

Central Carbon ◽

Pep Carboxykinase

ABSTRACT The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. colicells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

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