PdhR (pyruvate dehydrogenase complex regulator) controls the respiratory electron transport system in Escherichia coli

Abstract

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

FIG. 1.

PdhR-bound DNA fragments. Using genomic SELEX, PdhR-bound DNA fragments were isolated, the indicated four groups of which contained the promoter regions from the pdhR, cyoA, ndh, and yfiD genes. Open bars indicate the genomic DNA sequence, while thick bars indicate the regions of fragments isolated by SELEX. The dotted lines show the DNA fragments used for gel shift and DNase I footprinting assays. The vertical line shows the translation initiation site. Numbers on each line represent the distance (bp) from the respective initiation codon. Transcription initiation sites, shown by arrows, are from pdhR (25), cyoA (22), ndh (13), and yfiD (12).

FIG. 2.

Gel shift assay. (A) Fluorescently labeled DNA probes, each containing a segment of the pdhR, cyoA, yfiD, or ndh promoter (for the region of each probe, see Fig. 1), were incubated at 37°C for 30 min with the indicated amounts (0, 0.625, 1.25, 2.5, and 5 pmol) of PdhR (lanes 1 to 5) and directly subjected to PAGE. (B) Fluorescently labeled DNA probes of cyoA or ndh were incubated at 37°C for 30 min in the absence (lane 1) or presence (lanes 2 to 5) of 5 pmol of PdhR and in the absence (lane 2) or presence (lane 3, 100 mM; lane 4, 200 mM; lane 5, 500 mM) of increasing concentrations of pyruvate. Samples were subjected directly to PAGE.

FIG. 3.

DNase I footprinting of the ndh promoter. (Left) The fluorescently labeled ndh promoter (Fig. 2) was incubated with increasing amounts of purified PdhR (lane 1, 0 pmol; lane 2, 2.5 pmol; lane 3, 5 pmol; lane 4, 10 pmol) and subjected to DNase I footprinting assays. Lanes A, T, G, and C represent the sequence ladders. The black bar on the right indicates the PdhR-binding region. (Right) The PdhR-binding site on the ndh promoter, determined as described above, is indicated by a thick bar between positions −43 and −18. The transcription initiation site according to Green and Guest (13) is marked by an arrow. Recognition sequences for Fis, FNR, and IHF (boxed) were described previously by Jackson et al. (18), Meng et al. (21), and Green et al. (11), respectively.

FIG. 4.

DNase I footprinting of the cyoA promoter. (A) The fluorescently labeled cyoA promoter was incubated with increasing amounts of purified PdhR (lane 1, 0 pmol; lane 2, 2.5 pmol; lane 3, 5 pmol; lane 4, 10 pmol) and subjected to DNase I footprinting assays. Lanes A, T, G, and C represent sequence ladders. (B) The black bar (positions −185 to −158) on the right indicates the PdhR-binding region located upstream of the cyoA promoter. The nucleotide numbers represent the distance from the transcription initiation site, which was proposed previously by Minagawa et al. (22) and confirmed in this study. Recognition sequences for Fur, ArcA, and Fnr were predicted based on sequence analyses reported previously by Stojiljkovic et al. (35), Shalel-Levanon et al. (29), and Salmon et al. (28), respectively. Two CRP-binding sequences, CRP1 and CRP2, were proposed previously, but at different positions, by Minagawa et al. (22) (dotted lines) and Zheng et al. (40) (solid line). (C) Primer extension assay of the transcription initiation site of the cyoA operon in both wild-type KP7600 and its pdhR disruptant, JD20368, was performed using a fluorescently labeled probe. Data for the site identified thus agreed with those reported previously by Minagawa et al. (22).

FIG. 5.

Consensus sequence for the PdhR box. The PdhR-binding sites are derived from the ndh and cyoA promoters determined in this study, the previously reported pdhR promoter (25), and the predicted yfiD promoter (36). After searching the PdhR-box-like sequence along the entire E. coli genome, three additional targets have been identified: ybaJ, with the sequence (480189)ATTGGTgacACtAAT(480203); hemL or clcA, with the sequence (175079)ATTNGTNNNACCAAT(175093); and lldP, with the sequence (3863230)ATTGGNNNNACCAAT(3863216).

FIG. 6.

Effect of PdhR on the ndh and cyoABCD promoters. (A and B) The promoter assay vectors containing the ndh or cyoABCD promoters were transformed into wild-type (KP7600) and pdhR deletion mutant (JD20368) cells and grown in LB medium under aerobic (open bars) and anaerobic (closed bars) conditions. Promoter activities were determined at a cell density of an OD₆₀₀ of 0.6 and are represented as GFP/RFP ratios. (C and D) Wild-type cells and pdhR mutant transformants were grown in LB in the absence (lanes 1 and 2) or presence of 10 mM glucose (lanes 3 and 4) or 40 mM pyruvate (lanes 5 and 6) and under the aerobic (open bars) and anaerobic (closed bars) conditions. Promoter activities were determined at a cell density of an OD₆₀₀ of 0.2. (E) The activity of a single-copy cyoA promoter-lacZ fusion in wild-type (KP74201) (lane 1) and pdhR mutant (KP74202) (lane 2) cells was determined by measuring β-galactosidase activity at a cell density of an OD₆₀₀ of 0.6 under aerobic conditions.

FIG. 7.

Role of PdhR as a master regulator of the metabolic pathways including both NAD-linked oxidative decarboxylation of pyruvate to acetyl-CoA and respiratory electron transport of NADH to oxygen.