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Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli

Barrie Cassey , John R Guest , Margaret M Attwood
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb12878.x 325-329 First published online: 1 February 1998


The effects of changing environmental conditions on expression of the pdh operon were studied in strains containing pyruvate dehydrogenase (PDH) complexes having either one or three lipoyl domains per lipoate acetyltransferase chain. The expression of the pdh operon was lowered during growth on reduced carbon sources and when the mode of energy generation was changed from aerobic respiration to anaerobic respiration and fermentation. In contrast, growth at non-optimal pH increased expression. Operon expression was generally higher in the 1 lip strain compared to the 3 lip strain. Expression of the pdh operon was shown to be tightly controlled in response to environmental stimuli, consistent with its importance in defining metabolic flux.

Key words
  • Pyruvate metabolism
  • Pyruvate dehydrogenase complex
  • Gene expression
  • Environmental control
  • Redox
  • Escherichia coli

1 Introduction

In Escherichia coli, pyruvate is metabolised primarily by the pyruvate dehydrogenase (PDH) complex during aerobic growth, pyruvate formate-lyase (PFL) being increasingly used under anaerobic conditions [13]. The PDH complex catalyses the NAD+-linked oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. The E. coli complex contains multiple copies of three enzymatic subunits: pyruvate dehydrogenase (E1p); lipoate acetyltransferase (E2p); and lipoamide dehydrogenase (E3); expressed from the pdh operon, pdhR-aceEF-lpdA[1]. Synthesis of the complex is controlled at the transcriptional level by the pyruvate-responsive autoregulator, PdhR, which binds immediately downstream of the pdh promoter to repress transcription in the absence of pyruvate [4]. The PDH complex is also controlled at the enzyme level due to its inhibition by acetyl-CoA [5] and the NADH-sensitivity of the E3 subunit [6]. In E. coli, the E2p subunits have three lipoyl domains joined by flexible linkers which allow them to fulfil their role in active-site coupling within the assembled complex [2]. Genetically engineered strains with fewer than three lipoyl domains per E2p chain have been shown to be metabolically disadvantaged relative to the wild-type strain [2, 7]. Here, two strains with λpdhR-aceE′-lacZ reporter prophages, were used to investigate the effects of various environmental stimuli on pdh operon expression in strains that synthesise PDH complexes having only one or three lipoyl domains per E2p chain.

2 Materials and methods

2.1 Bacteria, phages and culture methods

Two isogenic derivatives of E. coli W3110 (prototroph) encoding PDH complexes having one lipoyl domain (JRG2931, 1 lip) or three (JRG2933, 3 lip) per E2p subunit [7] were used. The corresponding monolysogens, JRG3059 (1 lip, λG239) and JRG3060 (3 lip, λG239), each contain a λpdhR-aceE′-lacZ translational fusion prophage, used to monitor pdh expression [1]. The lysogens could not be grown in continuous culture due to prophage instability.

The minimal medium [7] for batch and chemostat cultures (500 and 700 ml, respectively) was supplemented with different carbon sources (mM): pyruvate, 40; gluconate, 20; glucose, 20; or mannitol, 20. Temperature (37°C), pH (value±0.1), and dissolved oxygen (>50% air saturation for aerobic cultures) were controlled [7]. Anaerobic cultures were established aerobically and allowed to become anaerobic by removing the air supply and later sparging with nitrogen gas (500 ml min−1) at a constant agitator speed (400 rpm). Inocula (50 ml) were grown aerobically for 16 h in the medium required for each experiment.

2.2 β-Galactosidase assay and measurement of NADH/NAD+ ratios

Samples (30–35 ml) from exponential-phase batch cultures were harvested (4000 rpm, 15 min), washed in buffer (K2HPO4, 40 mM; MgCl2, 4 mM), and the bacterial pellets stored at −20°C for subsequent and ultrasonic disruption at 4°C in 0.5 ml of the same buffer [7]. β-Galactosidase activity (ΔA420 h−1 mg protein−1 in cell-free extract) was measured [8] using a Labsystems iEMS plate-reader with a flat-bottomed microtitre plate and protein was assayed by the Bio-Rad procedure with γ-globulin as standard. Specific activities were averaged from triplicate samples from two or more independent cultures. Nucleotides were extracted and assayed [9] using flash-frozen samples (5 ml, in liquid N2) taken from high density steady-state carbon-limited cultures. The quoted ratios are averages from two independent samples.

3 Results and discussion

3.1 Effects of carbon source on pdh expression

Growth of E. coli in media containing different carbon sources leads to changes in cellular redox state [10], cell metabolism and yield [11]. The effects of carbon source on pdh expression in E. coli were studied with aerobic batch cultures of two pdhR-aceE′-lacZ reporter strains, JRG3059 (1 lip) and JRG3060 (3 lip), grown at a constant pH 7.0 with pyruvate, gluconate, glucose or mannitol as sole carbon and energy sources. The substrates were chosen to represent different oxido-reduction states (gluconate, glucose, mannitol) or the capacity to relieve transcription repression by PdhR at the pdh promoter (pyruvate) [4]. The β-galactosidase activities reflect combined changes in pdh transcription and translation (Fig. 1a) and as expected, they were highest during growth on pyruvate. With the other substrates, a direct relationship was observed between the level of pdh expression and the oxido-reduction state of the substrate, even though the differences in theoretical energy yields are relatively small for aerobic catabolism. Expression of the pdh operon was consistently higher in the 1 lip strain. The NADH/NAD+ ratios in JRG2933 (3 lip non-lysogenic parent of JRG3060) confirmed that there is a relationship between PDH complex synthesis and increasing intracellular NADH/NAD+ ratio, gluconate<glucose<mannitol (Fig. 1d). The high ratios in pyruvate-grown cultures may be due to an increase in carbon flux through the PDH complex stemming from the induction of pdh expression by pyruvate. It would also appear that the high NADH/NAD+ ratios associated with some substrates repress pdh expression, directly or indirectly.


The effects of environmental changes on pdh expression and NADH/NAD+ ratio. Expression of the pdh operon is indicated by the β-galactosidase activities (ΔA420 h−1 mg protein−1) of mid-exponential-phase batch cultures of strains containing a pdh-lacZ reporter fusion, □ JRG3059 (1 lip), and ▪ JRG3060 (3 lip), during growth: a: with different carbon sources; b: with different terminal electron acceptors, oxygen (dO2>50%), nitrate (100 mM), ferricyanide (50 mM), fumarate (50 mM), and none added (fermentation); and c: with glucose at different external pH values. The NADH/NAD+ ratios are shown for carbon-limited chemostat cultures of JRG2933 (3 lip) grown: d: with different substrates at pH 7; and e: with limiting glucose at different pH values.

3.2 Effects of terminal electron acceptor on pdh expression

E. coli can use different modes of energy transduction to support growth, so in the absence of oxygen, aerobic respiration is replaced by anaerobic respiration with alternative terminal electron acceptors, or by fermentation. The reporter strains were accordingly grown in controlled batch cultures with glucose and electron acceptors having significantly different electrochemical mid-point potentials (Em, mV): oxygen (+815); nitrate (+420); ferricyanide (+360); fumarate (+30); and none (−412, endogenous acetyl). With some exceptions, pdh expression was highest under aerobic conditions, falling with decreasing electron acceptor potential to low levels during fumarate respiration and fermentation, where the complex is probably inactive (Fig. 1b). This suggests that the efficiency of energy generation (and hence nucleotide recycling) influences pdh expression. Expression of the pdh operon was generally higher in the 1 lip strain, particularly during nitrate respiration, where expression in this strain greatly exceeded that observed under any other condition. During nitrate respiration the PDH complex and pyruvate formate-lyase are both active, but at sub-maximal levels [3]. Presumably, the 1 lip-PDH complex has to be expressed at greatly elevated levels in order to meet the required carbon flux to acetyl-CoA. During fermentation, the pdh operon was still expressed (Fig. 1b). This is consistent with the lower but continued synthesis of PDH complex subunits during fermentative growth [12].

3.3 Effects of pH on pdh expression

The effects of pH were examined using controlled batch cultures growing with glucose at pH 6, 7, and 8 (Fig. 1c). Expression of the pdh operon increased as the pH diverged from the optimum value, and the effects were again more apparent in the 1 lip strain. The intracellular NADH/NAD+ ratios of glucose-limited cultures of JRG2933 (3 lip non-lysogenic parental strain) increased as the environmental pH increased from 6.0 to 8.0 (Fig. 1e). A similar trend has been observed with anaerobic cultures of Enterococcus faecalis[9].

The current studies with pdh-lacZ reporters show that pdh expression is modulated (in vivo) in response to the environment. The results, which reflect changes in transcription and translation rather than enzymatic activity, indicate that pdh expression is higher in the 1 lip strain than in the 3 lip strain under all conditions except fumarate respiration and fermentation (where the PDH complex is not essential). Presumably, pdh expression could be increased in the 1 lip strain, via pyruvate induction, in an attempt to compensate for the lower efficiency of carbon flux through the modified PDH complex [7]. Also, in the wild-type strain, it would appear that pdh expression is repressed under conditions of NADH (and acetyl-CoA/acetyl phosphate) accumulation. This could be mediated by ArcA, which represses in response to such stimuli, or via a potentiation of PdhR-mediated repression by the same stimuli. The role of ArcA in regulating PDH complex synthesis is uncertain because although anaerobic PDH complex activity is increased 1.7-fold (back to the aerobic level) in an arcA mutant [13], the pdh promoter appears not to be repressed by ArcA in studies with lacZ fusions [1].


This work was supported by a project grant and studentship from the Biotechnology Directorate of the Biotechnology and Biological Sciences Research Council.


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