Abstract
BackgroundThe pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach.Results22 new genes contained in two operons controlled by PdhR (previously only 20 regulatory targets in eight operons were known) were identified by analysing a large-scale dataset of E. coli from the Many Microbes Microarray Database and novel expression data from a pdhR knockout strain, as well as a PdhR overproducing strain. We identified a regulation of the glycolate utilization operon glcDEFGBA using chromatin immunoprecipitation and gel shift assays. We show that this regulation could be part of a cross-induction between genes necessary for acetate and pyruvate utilisation controlled through PdhR. Moreover, a link of PdhR regulation to the replication machinery of the cell via control of the transcription of the dcw-cluster was verified in experiments. This augments our knowledge of the functions of the PdhR-regulon and demonstrates its central importance for further cellular processes in E. coli.ConclusionsWe extended the PdhR regulon by 22 new genes contained in two operons and validated the regulation of the glcDEFGBA operon for glycolate utilisation and the dcw-cluster for cell division proteins experimentally. Our results provide, for the first time, a plausible regulatory link between the nutritional status of the cell and cell replication mediated by PdhR.
Highlights
The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes
Using chromatin immunoprecipitation (ChIP) in combination with quantitative PCR and gel shift assays we discovered that the glcDEFGBA operon as well as the mraZW-ftsLImurEF-mraY-murD-ftsW-murGC-ddlB-ftsQAZ-lpxC transcription unit are controlled by pyruvate dehydrogenase complex regulator (PdhR)
The applied growth conditions in complex rich medium might not be suitable for the detection of all PdhR-DNA interactions, the overproduction of PdhRhis should facilitate binding, to DNA fragments which might be bound with low affinity
Summary
The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. The pyruvate dehydrogenase complex of Escherichia coli is encoded by the operon pdhR-aceE-aceF-lpdA. The first gene encodes the pyruvate dehydrogenase complex regulator (PdhR), which functions as a transcriptional regulator in a self-regulatory manner for this operon [1]. The multienzyme complex of the pyruvate dehydrogenase complex consists of 24 subunits of the pyruvate dehydrogenase (aceE), 24 subunits of the dehydrolipoate acetyltransferase (aceF), and 12 subunits of the dehydrolipoamide dehydrogenase (lpdA). The tomB-hha operon (antitoxin (TomB)-toxin (Hha)-module) [4] and the genes hemL (glutamate-1-semialdehyde aminotransferase) [4], yfiD (pyruvate formate-lyase subunit) [6], and lipA (lipoate synthase) [7] are directly controlled by PdhR
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