1 Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal
2 Centro de Investigaciones Biológicas, Velazquez 144, Madrid, Spain
Correspondence
Helena Santos
santos{at}itqb.unl.pt
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycolytic regulation in L. lactis has been related to the high fructose 1,6-bisphosphate (FBP) concentration that activates lactate dehydrogenase (LDH) and pyruvate kinase (PK), directing the flux towards the production of lactate. Accordingly, accumulation of phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3-PGA) in starved cells is regarded as a consequence of PK inhibition by high inorganic phosphate (Pi) and low FBP concentrations associated with sugar depletion (Mason et al., 1981; Thompson, 1987
). Recent studies have questioned the role of FBP and postulated that inhibition or activation exerted by the cellular NADH/NAD+ ratio on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or LDH is the main issue regulating glycolysis and the shift from homo- to heterolactic fermentation (Garrigues et al., 1997
). On the other hand, based on non-invasive NMR measurements of NAD+ and NADH, we have shown that the glycolytic flux in resting cells is not appreciably influenced by the NADH/NAD+ ratio (Neves et al., 2002b
). Recently, using an approach that involves manipulation of enzyme activities in small steps above and below the wild-type level and metabolic control analysis, Jensen and collaborators have shown that GAPDH has no control over glycolytic flux (Solem et al., 2003
). Similarly, LDH and also phosphofructokinase (PFK) have been ruled out as major sites of control of lactate production in L. lactis (Andersen et al., 2001b
; Koebmann et al., 2002b
). Hence, the control of glycolysis in L. lactis remains elusive.
PK (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) catalyses the last step of glycolysis, where the phosphoryl group of PEP is transferred to ADP to form pyruvate and ATP. The reaction is irreversible under physiological conditions and has long been considered as critical for the regulation of metabolic flux in the second part of glycolysis. This central position is evident from the regulatory properties of PK, a typical allosteric protein (Valentini et al., 2000). The lactococcal enzyme has been purified and characterized (Crow & Pritchard, 1976
, 1982
). It is activated by FBP, the activity being a sigmoidal function of the activator concentration, and the affinity for PEP and ADP is also enhanced by FBP. Furthermore, the Ka for FBP increases drastically in the presence of low concentrations of Pi. The magnitude of the FBP and Pi pools in L. lactis has been assessed in several studies; FBP accumulates up to 50 mM in the presence of glucose or lactose, whereas high concentrations of Pi, 3-PGA, 2-PGA and PEP are typical of starved cells (Thompson & Thomas, 1977
; Thompson & Torchia, 1984
; Neves et al., 1999
, 2002b
).
Early studies provided evidence for a pivotal regulatory role of PK in the glycolytic metabolism of L. lactis (Mason et al., 1981; Thompson & Torchia, 1984
). These data prompted us to evaluate the effect of PK overproduction on the physiology of this model organism and to investigate the role of this enzyme in the regulation of the overall glycolytic flux. NMR techniques were used to monitor the kinetics of the intracellular pools of several glycolytic metabolites, NAD+, NADH, ATP and Pi in living cells of L. lactis overproducing PK.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial strains and media.
Escherichia coli MC1061 was grown in LuriaBertani broth with aeration at 37 °C. Lactococcus lactis strains MG1363 (Gasson, 1983), NZ9000 (expressing the nisRK regulatory genes; Kuipers et al., 1998
) and its derivatives were routinely cultivated without aeration in M17 medium supplemented with 1 % (w/v) glucose. Chloramphenicol was used at a concentration of 20 or 5 µg ml1 for selection of pNZ8020-based plasmids (de Ruyter et al., 1996
) in E. coli or L. lactis, respectively. Bacterial growth was monitored spectrophotometrically at 600 nm.
DNA techniques and transformation.
All manipulations with recombinant DNA in E. coli MC1061 (used as an intermediate host for cloning) were carried out according to standard procedures (Sambrook et al., 1989) and as specified by the enzyme manufacturers. Chromosomal and plasmid DNA of L. lactis was isolated as described by Leenhouts et al. (1989)
and O'Sullivan & Klaenhammer (1993)
, respectively. PCR reactions were performed in a total volume of 100 µl with 10 mM Tris/HCl (pH 8·8), 2 mM MgCl2, 50 mM NaCl, 200 µM each deoxyribonucleoside triphosphate, 1 U DyNAzyme, 50 pmol of each primer and 1 µg template DNA obtained from MG1363. Amplification was performed in 25 cycles, each cycle consisting of a denaturation step at 95 °C for 1 min, a primer annealing step at 55 °C for 1 min, and a primer extension step at 72 °C for 2 min. Preparation of competent L. lactis cells and electrotransformation was performed as described by Dornan & Collins (1990)
.
Construction of plasmids and mutant strains.
Primers Pyk1 (5'-GCGCTGGATCCCGCATAATTAGGGG-3') and Pyk2 (5'-CGTATGAATTCGCCTGTCGATAGCGG-3'), based on the sequence of the PK gene (accession no. LO7920) published previously (Llanos et al., 1993), were designed to introduce BamHI and EcoRI restriction sites (underlined in the primer sequences) upstream and downstream, respectively, of the pyk gene coding region. A PCR product with the expected size (1·6 kb) was obtained and cloned as a BamHIEcoRI fragment into the similarly digested, high-copy-number shuttle vector pNZ8020 under control of the nisA promoter. The ligation mixture was transformed into E. coli MC1061, and a recombinant plasmid carrying the appropriate insert (designated pNZpyk) was selected. The pNZpyk and pNZ8020 plasmids were independently introduced by electroporation to L. lactis NZ9000 for further studies.
Fermentation under controlled conditions.
L. lactis cultures of NZ9000(pNZpyk) and NZ9000(pNZ8020) were grown at 30 °C in a 2 litre fermenter in the defined medium (CDM) described by Poolman & Konings (1988), containing 1 % (w/v) glucose, 5 µg chloramphenicol ml1 and 1 ng nisin ml1. Strain NZ9000 was grown in the same medium without addition of chloramphenicol and nisin. For the measurements of pyridine nucleotide pools, [5-13C]nicotinic acid (5 mg l1), synthesized as described before (Neves et al., 2002a
), was supplied, and aspartate and asparagine were omitted. The pH was kept at 6·5 by automatic addition of 10 M NaOH and a low agitation rate (70 r.p.m.) was used to keep the fermentation broth homogeneous. Anaerobic conditions were established by gassing the medium with argon for 2 h before inoculation (4 % inoculum with a culture grown overnight in the same medium); for aerobic growth, the dissolved oxygen was monitored with an oxygen electrode (Ingold). A specific air tension of 90 % was maintained by automatic control of the airflow. Growth was evaluated by measuring the turbidity of the samples at 600 nm (OD600) and calibration against cell dry weight measurements.
Preparation of ethanol extracts for analysis by 31P-NMR and quantification of phosphorylated metabolites.
Ethanol extracts were prepared from culture samples (approx. 70 mg protein for each extract) obtained during growth as previously described (Ramos et al., 2001), and the concentration of intracellular metabolites was calculated from the peak areas in 31P spectra by comparing with the area of the resonance due to methylphosphonic acid added as internal standard. Assignment of resonances was achieved by addition of the pure compounds to the extracts or by comparison with previous studies (Ramos & Santos, 1996
; Ramos et al., 2001
).
In vivo NMR experiments.
Cells were harvested in the mid-exponential phase (OD600 2·2), centrifuged and suspended in 50 mM potassium phosphate (KPi) buffer, pH 6·5, to a protein concentration of approximately 13 mg ml1. In vivo NMR experiments were performed under anaerobic (argon atmosphere) or aerobic conditions (oxygen saturation) as described previously (Neves et al., 1999). [1-13C]Glucose (or [6-13C]glucose) was added and the time-course for its consumption, product formation, and build-up and consumption of intracellular metabolite pools was monitored. After glucose exhaustion, and when no changes in the resonances due to end products and intracellular metabolites were observed, a cell extract was prepared (Neves et al., 1999
): briefly, an aliquot of the cell suspension was passed through a French press (twice at 120 MPa); the resulting extract was incubated at 80 °C for 15 min in a sealed tube, cooled on ice; the cell debris was removed by centrifugation (30 000 g, 10 min, 4 °C) and the supernatant (denoted hereafter as total NMR-sample extract) was stored at 20 °C.
An aliquot of the cell suspension used for in vivo NMR was saved to prepare a crude extract and measure the level of PK overproduction. Protein concentration in cell suspensions was determined by the Lowry method using bovine serum albumin as a standard after disruption of cells by treatment with 1 M NaOH and incubation at 80 °C for 5 min.
Quantification of end products.
Lactate, acetate and formate were quantified in total NMR sample extracts by 1H-NMR as described elsewhere (Neves et al., 1999). The concentrations of metabolic intermediates and minor end products that remained inside the cells (PEP and 3-PGA) were determined by 13C-NMR. The concentration of labelled lactate determined by 1H-NMR was used as a standard to calculate the concentration of the other metabolites in the sample. Carbon balances from glucose were always above 95 % after taking into account both extracellular and intracellular metabolites.
Quantification of intracellular metabolites in living cells by 13C-NMR.
Due to the fast pulsing conditions used for acquiring in vivo 13C-spectra, there is no direct correlation between concentrations and peak intensities. The correction factors that allowed the conversion of peak areas of glucose 6-phosphate (G6P), FBP, 3-PGA, NAD+ and NADH into intracellular concentrations were determined as described previously (Neves et al., 1999, 2002a
). The quantitative kinetic data for intracellular metabolites were calculated from their peak areas by applying the correction factors and comparing with the intensity of the lactate resonance in the last spectrum of the sequence. Intracellular metabolite concentrations were calculated using a value of 2·9 ml (mg protein)1 for the intracellular volume (Poolman et al., 1987
).
NMR spectroscopy.
All NMR experiments with living cells were run at 30 °C with a quadruple-nucleus probe head on a Bruker DRX500 spectrometer as described before (Neves et al., 1999, 2002b
). Although individual experiments are illustrated in each figure, each type of in vivo NMR experiment was repeated at least twice and the results were highly reproducible. The values reported are means of two or three experiments and the accuracy varied from 2 % (for end products) to 15 % (for intracellular metabolites with concentrations below 5 mM). The concentration limit for detection of intracellular metabolites in living cells was around 0·6 mM in the experiments where the pools of nicotinamide nucleotides were monitored (2·2 min acquisition time for each spectrum), and 34 mM in the remaining experiments (30 s acquisition).
Quantification of the pools of phosphorylated metabolites in growing cells and measurements of PK activity by 31P-NMR were performed as described previously (Ramos et al., 2001). Carbon and phosphorus chemical shifts were referenced to the resonances of external methanol or 85 % H3PO4 designated at 49·3 p.p.m. and 0·0 p.p.m., respectively.
Preparation of crude cell extracts for determination of enzyme activities.
Cells were cultivated in defined medium as described above until mid-exponential growth phase, harvested, washed and suspended in 50 mM MOPS buffer pH 6·5. Cell extracts were prepared by mechanical disruption using a French press (three passages at 120 MPa), debris was removed by centrifugation (30 000 g, 20 min, 4 °C), and the extracts were used immediately for determination of enzyme activity. The protein concentration in crude cell extracts was measured by the method of Bradford (1976) using bovine serum albumin as a standard.
Purification of PK.
PK was purified to electrophoretic homogeneity from approximately 65 g (wet wt) of cells of the NZ9000(pNZpyk) strain essentially as described by Crow & Pritchard (1976).
Enzyme assays.
PK was routinely assayed in cell extracts by the spectrophotometric method described by Crow & Pritchard (1976). LDH, NADH oxidase (Garrigues et al., 1997
) and PFK (Fordyce et al., 1982
) were measured as described before.
The effect of activators and inhibitors of PK was determined by 31P-NMR spectroscopy using the purified enzyme. The 3 ml reaction mixtures contained 100 mM Tris/HCl buffer pH 7·5, 5 mM MgCl2, 10 mM KCl, 5 mM FBP, 2 mM ADP and 50 µg pure enzyme. The reactions were started by addition of 2 mM PEP and the time-course for its consumption was monitored; the effect of Pi, FBP, NADH, NAD+, 3-PGA and ATP on the activity of PK was studied by adding these compounds to the reaction mixtures. The reactions were stopped before PEP exhaustion by incubation at 80 °C for 5 min and the rate of PEP consumption was calculated by comparison of the area of the PEP resonance to that of a known amount of methylphosphonate added as an internal standard. One unit of enzyme activity was defined as the amount of enzyme catalysing the conversion of 1 µmol PEP min1 under the experimental conditions used.
All the determinations were made at least in triplicate in two extracts obtained from independent cultures.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pools of glycolytic intermediates monitored by in vivo NMR
Strains NZ9000(pNZpyk) and NZ9000(pNZ8020) were grown in defined medium containing 1 ng nisin ml1, harvested in the mid-exponential phase, and glucose metabolism was studied in vivo by NMR. Lactate production, glucose consumption and glycolytic intermediates during the metabolism of [1-13C]glucose (40 mM) in both strains under an argon atmosphere are shown in Fig. 1(a, b). The conversion of glucose was homofermentative in strains NZ9000(pNZpyk) and NZ9000(pNZ8020), the glucose consumption rates being 0·19±0·01 and 0·22±0·02 µmol min1 (mg protein)1, respectively. The reduction of the glucose consumption rate in NZ9000(pNZpyk) when compared to the reference strain NZ9000(pNZ8020) is noteworthy; a similar trend was observed in a L. lactis construct overproducing NADH oxidase under the control of the nisin promoter (Neves et al., 2002a
). A striking difference in the profile of depletion of the FBP pool was found. Although the maximal intracellular concentration reached (approx. 45 mM) was similar in both strains examined, the consumption of FBP after glucose exhaustion proceeded at a much higher rate in the PK-overproducing strain. In addition, 3-PGA accumulated to a maximal concentration of 10 mM in NZ9000(pNZ8020), whereas in NZ9000(pNZpyk) this metabolite was always below the in vivo detection limit (Fig. 1a, b
). It is worth mentioning that 3-PGA was also not visible in the 13C-NMR spectrum of the NMR-sample extract (not shown) obtained as described in Methods, meaning that its intracellular concentration was below 1·8 mM (detection limit). This result was due to the overproduction of PK in NZ9000(pNZpyk) since this strain behaved like NZ9000(pNZ8020) with respect to the pattern of FBP and 3-PGA accumulation when intermediate levels of PK (7 units mg1) were achieved by lowering the concentration of nisin to 0·25 ng ml1 (data not shown).
|
Fig. 2 shows the data obtained during the metabolism of [6-13C]glucose (40 mM) by both strains under aerobic (oxygen-saturated) atmosphere. The use of glucose labelled on C-6 allowed us to detect and quantify the resonance of G6P, which otherwise was obscured by the intense peaks due to [1-13C]glucose. The rates of glucose consumption by NZ9000(pNZpyk) and NZ9000(pNZ8020) were 0·12±0·01 and 0·16±0·01 µmol min1 (mg protein)1, respectively. Lowered glycolytic fluxes have been consistently observed under aerobic conditions in several L. lactis strains (Neves et al., 2002a
). The behaviour of strain NZ9000(pNZ8020) (Fig. 2b, d
) was comparable to that reported for MG1363 under an oxygen atmosphere (Neves et al., 2002b
): after the addition of glucose, FBP and G6P rose to approximately 48 and 5 mM, respectively, decreasing to undetectable levels after glucose depletion, whilst 3-PGA and PEP accumulated to concentrations of approximately 32 and 10 mM, respectively, levels that are much higher than those measured under an argon atmosphere. Acetate (7·8 mM) and acetoin (1·1 mM) were produced in addition to lactate (67·2 mM). In the NZ9000(pNZpyk) strain (Fig. 2a, c
), the production of acetate and acetoin was, respectively, two- and eightfold higher than in NZ9000(pNZ8020). 3-PGA and PEP were not detected, either in vivo or in NMR-sample extracts. The acceleration of the FBP consumption rate occurred in both strains upon switching from anaerobic to aerobic conditions (compare Figs 1 and 2
), but the maximal FBP level (approx. 37 mM) in the NZ9000(pNZpyk) strain was clearly lower under an oxygen atmosphere as compared to anaerobic conditions (approx. 45 mM).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present work takes advantage of a similar experimental approach to provide a detailed picture of glycolysis in a strain overproducing PK, a key glycolytic enzyme assumed to play a major role in the regulation of glycolysis. The most notable consequences of the overproduction of PK were the acceleration of the rates of FBP depletion and NAD+ recovery once glucose was exhausted, and the lack of accumulation of 3-PGA and PEP, the two metabolites associated with cell starvation. In strain NZ9000(pNZ8020), the level of FBP decreased very slowly after reaching the intracellular level of about 20 mM, a profile identical to that of strain MG1363, the parent strain of NZ9000 (Neves et al., 2002b). This observation denotes a constriction at the level of PK, further evidenced by the accumulation of metabolites immediately upstream of pyruvate, namely 3-PGA and PEP (Neves et al., 2002a
). The higher rates of FBP depletion and of NAD+ recovery, and the absence of detectable pools of 3-PGA or PEP in the PK-overproducing strain, suggest the occurrence of a metabolic bottleneck at the level of PK in NZ9000(pNZ8020) when glucose became limiting; in the PK overproducer this constriction was no longer present, allowing, in addition to a rapid FBP consumption, a complete carbon flow to lactate, thus preventing accumulation of 3-PGA and PEP. The obstruction at the level of PK is primarily ascribed to accumulation of Pi, a well-known inhibitor of the enzyme, counteracting the activating properties of FBP (Crow & Pritchard, 1976
, 1982
). In fact, when glucose transport stopped, the concentration of Pi rose abruptly due to the release of phosphate engaged mainly in the large FBP pool and other phosphorylated metabolites (Fig. 1
). The data provide clear evidence for the role of Pi as an important regulator of the flux through PK in vivo.
The absence of 3-PGA and PEP (below the detection limit of the technique) is even more striking when aerobic conditions are considered (Fig. 2c) since this and earlier studies have shown that the levels of these metabolites are greatly increased in the presence of oxygen (Fig. 1b
versus Fig. 2d
and Neves et al., 2002a
). In strain NZ9000(pNZ8020) approximately 50 % of the FBP pool was converted to 3-PGA and PEP (Fig. 2d
), meaning that the amount of carbon processed via PK, once glucose was depleted, was low. However, this bottleneck at the level of PK was completely overcome by 15-fold overproduction of PK (Fig. 2c
).
It has been shown that the NAD+ pool is maintained at the maximum level in MG1363 when glucose is actively metabolized (Neves et al., 2002b). Surprisingly, the level of NAD+ decreased gradually during glucose consumption in the PK overproducer, indicating that NADH reoxidation was less efficient in this strain. We think that this is caused by the depression of the LDH activity in NZ9000(pNZpyk), an unexpected lateral effect of the genetic manipulation strategy pursued here. One could speculate that the nisin induction system exerts a deleterious effect on the general transcription machinery and indirectly affects expression of the pfkpykldh (las) operon. However, this explanation is not satisfactory since, at least under anaerobic conditions, the PK and LDH activities of NZ9000 were not appreciably affected by the presence of the vector plasmid. In addition, the low levels of LDH in strain NZ9000(pNZpyk) could not be due to downregulation of transcription of the las operon mediated by PK overexpression, since the levels of PFK were similar in both engineered strains used in the present work (Table 1
). Surprisingly, Andersen et al. (2001a)
observed that alterations of the las promoter affect differentially the expression of the three genes of the operon. One explanation could be that the las operon is subjected to posttranscriptional regulation, which results in alterations of its mRNA fate and consequently modulates the translation efficiency of its genes. This view is supported by the observation of processed species of the las mRNA (Luesink et al., 1998
). If this is the case, the presence of chloramphenicol (a translation inhibitor) during growth of cultures carrying plasmids could differentially affect expression of pfk and ldh. Moreover, the pronounced inhibitory effect in the synthesis of LDH could explain the lower growth rate of NZ9000(pNZpyk) than that of NZ9000(pNZ8020).
The level of LDH in NZ9000(pNZpyk) was reduced to approximately 50 % of that of NZ9000, and this may be sufficient to create a bottleneck at the level of LDH and hinder the regeneration of NAD+. In fact, according to Andersen et al. (2001b), L. lactis has limited excess capacity of LDH, only 70 % more than needed to sustain the lactate flux in the wild-type cells. Interestingly, despite the lower NAD+/NADH ratio observed in the PK-overproducing strain, glucose was metabolized largely to lactate as in the wild-type strain. Therefore, it seems that the redistribution of carbon flux in the pyruvate node does not depend critically on the level of NAD+, and the NAD+/NADH ratio can vary to some extent without causing noticeable changes in the composition of end products.
The decreased LDH activity was probably also the main reason for the shift towards acetate and acetoin production, under aerobic conditions. The production of acetate and acetoin was considerably higher in the PK-overproducer than in NZ9000(pNZ8020), since the activity of LDH was approximately threefold lower in the former strain while NADH oxidase activity was similar in both strains.
Overexpression of the gene encoding PK, a postulated regulatory enzyme, did not lead to an increased glycolytic flux in non-growing cells, instead a slight decrease was observed; also, the growth rate of the PK-overproducer was reduced (17 %). It is worth mentioning that the patterns of glycolytic intermediates in growing cells (Fig. 4) were similar to those measured by in vivo NMR in non-growing cell suspensions, namely the absence of 3-PGA in the PK-overproducer after glucose depletion. Lower growth and glucose consumption rates in this mutant may be a consequence of (i) diminished level of LDH, and/or (ii) protein burden, as observed in Zymomonas mobilis (Snoep et al., 1995
) and E. coli, where overproduction of PK or PFK led to reduced glucose bioconversion rates relative to the control (Emmerling et al., 1999
).
A recent study using metabolic control analysis excluded a role in the control of flux for the enzymes encoded by the las operon in growing cells of L. lactis MG1363 (Koebmann et al., 2002b). The same laboratory has reported that the demand for ATP exerts a strong control on the glycolytic flux of resting cells (Koebmann et al., 2002a
). We found that the glycolytic flux in resting cells of L. lactis did not increase in response to a 15-fold increase in the level of PK, but when glucose became limiting, PK overproduction allowed a greater flux through PK and led to complete channelling of carbon to end products without accumulation of the glycolytic intermediates typical of starvation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersen, H. W., Pedersen, M. B., Hammer, K. & Jensen, P. R. (2001b). Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis. Eur J Biochem 268, 63796389.
Bailey, J. E. (2001). Reflections on the scope and the future of metabolic engineering and its connections to functional genomics and drug discovery. Metab Eng 3, 111114.[CrossRef][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Crow, V. L. & Pritchard, G. G. (1976). Purification and properties of pyruvate kinase from Streptococcus lactis. Biochim Biophys Acta 438, 90101.[Medline]
Crow, V. L. & Pritchard, G. G. (1982). Pyruvate kinase from Streptococcus lactis. Methods Enzymol 90, 165170.[Medline]
de Ruyter, P. G., Kuipers, O. P. & de Vos, W. M. (1996). Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62, 36623667.
Dornan, S. & Collins, M. A. (1990). High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230. Lett Appl Microbiol 11, 6264.[Medline]
Emmerling, M., Bailey, J. E. & Sauer, U. (1999). Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes. Metab Eng 1, 117127.[CrossRef][Medline]
Fordyce, A. M., Moore, C. H. & Pritchard, G. G. (1982). Phosphofructokinase from Streptococcus lactis. Methods Enzymol 90, 7782.[Medline]
Garrigues, C., Loubiere, P., Lindley, N. D. & Cocaign-Bousquet, M. (1997). Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. J Bacteriol 179, 52825287.[Abstract]
Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154, 19.[Medline]
Koebmann, B. J., Solem, C., Pedersen, M. B., Nilsson, D. & Jensen, P. R. (2002a). Expression of genes encoding F1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis. Appl Environ Microbiol 68, 42744282.
Koebmann, B. J., Andersen, H. W., Solem, C. & Jensen, P. R. (2002b). Experimental determination of control of glycolysis in Lactococcus lactis. Antonie van Leeuwenhoek 82, 237248.[CrossRef][Medline]
Kuipers, O. P., De Ruyter, P. G. G. A., Kleerebezem, M. & de Vos, W. M. (1998). Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64, 1521.[CrossRef]
Leenhouts, K. J., Kok, J. & Venema, G. (1989). Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55, 394400.[Medline]
Llanos, R. M., Harris, C. J., Hillier, A. J. & Davidson, B. E. (1993). Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase. J Bacteriol 175, 25412551.[Abstract]
Luesink, E. J., van Herpen, R. E., Grossiord, B. P., Kuipers, O. P. & de Vos, W. M. (1998). Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 30, 789798.[CrossRef][Medline]
Mason, P. W., Carbone, D. P., Cushman, R. A. & Waggoner, A. S. (1981). The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis. J Biol Chem 256, 18611866.
Neves, A. R., Ramos, A., Nunes, M. C., Kleerebezem, M., Hugenholtz, J., de Vos, W. M., Almeida, J. S. & Santos, H. (1999). In vivo NMR studies of glycolytic kinetics in Lactococcus lactis. Biotechnol Bioeng 64, 200212.[CrossRef][Medline]
Neves, A. R., Ramos, A., Costa, H., van Swam, I. I., Hugenholtz, J., Kleerebezem, M., de Vos, W. M. & Santos, H. (2002a). Effect of different NADH oxidase levels on glucose metabolism of Lactococcus lactis: kinetics of intracellular metabolite pools by in vivo NMR. Appl Environ Microbiol 68, 63326342.
Neves, A. R., Ventura, R., Mansour, N., Shearman, C., Gasson, M. J., Maycock, C., Ramos, A. & Santos, H. (2002b). Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD+ and NADH pools determined in vivo by 13C-NMR. J Biol Chem 277, 2808828098.
O'Sullivan, D. J. & Klaenhammer, T. D. (1993). Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59, 27302733.[Abstract]
Poolman, B. & Konings, W. N. (1988). Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. J Bacteriol 170, 700707.[Medline]
Poolman, B., Smid, E. J., Veldkamp, H. & Konings, W. N. (1987). Bioenergetic consequences of lactose starvation for continuously cultured Streptococcus cremoris. J Bacteriol 169, 14601468.[Medline]
Ramos, A. & Santos, H. (1996). Citrate and sugar cofermentation in Leuconostoc oenos, a 13C nuclear magnetic resonance study. Appl Environ Microbiol 62, 25772585.[Abstract]
Ramos, A., Boels, I. C., de Vos, W. M. & Santos, H. (2001). Regulation of carbon flux from glycolysis to exopolysaccharide biosynthesis in Lactococcus lactis. Appl Environ Microbiol 67, 3341.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Snoep, J. L., Yomano, L. P., Westerhoff, H. V. & Ingram, L. (1995). Protein burden in Zymomonas mobilis: negative flux and growth control due to overproduction of glycolytic enzymes. Microbiology 141, 23292337.
Solem, C., Koebmann, B. J. & Jensen, P. R. (2003). Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. J Bacteriol 185, 15641571.
Thompson, J. (1987). Regulation of sugar transport and metabolism in lactic acid bacteria. FEMS Microbiol Rev 46, 221231.[CrossRef]
Thompson, J. & Thomas, T. D. (1977). Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis. J Bacteriol 130, 583595.[Medline]
Thompson, J. & Torchia, D. A. (1984). Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis. J Bacteriol 158, 791800.[Medline]
Valentini, G., Chiarelli, L., Fortin, R., Speranza, M. L., Galizzi, A. & Mattevi, A. (2000). The allosteric regulation of pyruvate kinase. A site-directed mutagenesis study. J Biol Chem 275, 1814518152.
Received 8 August 2003;
revised 19 December 2003;
accepted 5 January 2004.