Division of Molecular and Genetic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, UK1
Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Sheffield S10 2TN, UK2
Author for correspondence: Jonathan G. Shaw. Tel: +44 114 271 3517. Fax: +44 114 273 9926. e-mail: J.G.Shaw{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: nuclear magnetic resonance, phosphotransacetylaseacetate kinase pathway, proton NMR, carbon-13 NMR
Abbreviations: ACK, acetate kinase; AP5A, P1,P5-di(adenosine-5) pentaphosphate; CAC, citric acid cycle; CFE, cell-free extract; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PTA, phosphotransacetylase; TSP, trimethylsilyl propionate
a Present address: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
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INTRODUCTION |
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Many recent investigations of N. meningitidis have concentrated on a number of potential virulence factors. Very little is known about the possible links between metabolic processes and pathogenic mechanisms which may lead to the onset of systemic infection. Growth of N. meningitidis requires pyruvate, lactate, or glucose as a sole carbon source, in addition to carbon dioxide (Chapin, 1918 ). N. meningitidis is biochemically similar to Neisseria gonorrhoeae, but very little work has been carried out into neisserial physiology over the past 20 years (Holten, 1974a
, b
; Jyssum, 1960
).
The study of the catabolism of glucose by N. meningitidis and N. gonorrhoeae has indicated the involvement of the EntnerDoudoroff pathway, the pentose phosphate pathway and the citric acid cycle (CAC) (Morse et al., 1979 ). Investigations concerning the catabolism of pyruvate by N. meningitidis and N. gonorrhoeae indicated involvement of the CAC (Holten, 1975
, 1976a
). A pyruvate dehydrogenase system is present in N. meningitidis, which converts pyruvate to acetyl-CoA and CO2 (Jyssum, 1960
). The acetyl-CoA is then more slowly catabolized via the CAC. Lactate is utilized via its conversion to pyruvate by at least three different meningococcal lactate dehydrogenase (LDH) enzymes (Erwin & Gotschlich, 1993
, 1996
). Studies of growth on lactate and glucose have been carried out with N. gonorrhoeae, which showed an accumulation of acetate in the medium; this is utilized to a limited extent when the primary carbon source has been depleted (Hebeler & Morse, 1976
; Morse et al., 1979
; Morse, 1979
).
The study of the CAC of Neisseria began with the work of Tonhazy & Pelczar (1953) . They investigated the rate of oxidation and total oxygen consumption of intermediates of the CAC in N. gonorrhoeae. This method of analysis only led to a tentative prediction of the likely order of utilization of the substrates. Jyssum (1960)
initiated studies on the CAC in N. meningitidis. The individual reactions of a number of enzymes were examined, although the extracts were prepared from plate-grown cells. A number of enzymes of the CAC were identified, including aconitase, isocitrate dehydrogenase, citrate synthase, 2-oxoglutarate dehydrogenase, succinate dehydrogenase, fumarase and a malic enzyme dependent on NADP. However, no specific activities for the majority of the enzymes were reported.
Hebeler & Morse (1976) carried out a more comprehensive study of the CAC in N. gonorrhoeae. From extracts of cells grown in conditions of glucose depletion, all the CAC enzymes were identified in addition to phosphoenolpyruvate (PEP) carboxylase, while the extracts from glucose-grown cells lacked activity for aconitase, isocitrate dehydrogenase, malate oxidase (membrane-bound malate dehydrogenase) and PEP carboxylase. PEP carboxylase activity was identified in N. meningitidis (Jyssum & Jyssum, 1961
). The presence of this enzyme was further identified in all strains of Neisseria examined (Holten & Jyssum, 1974
). Further investigations by Holten (1976b
) of the malate oxidase established that, unlike malate dehydrogenase, the oxidation of malate was pyridine nucleotide (NAD) independent. Non-pathogenic neisseriae express both a soluble NAD-dependent malate dehydrogenase and a FAD-dependent malate oxidase (Jyssum & Jyssum, 1961
).
The complete genome sequence of N. meningitidis Serogroup B strain MC58 (Tettelin et al., 2000 ) suggests that a complete CAC may be present. In this study we provide firm biochemical evidence to support the predictions from the genome sequence. The central metabolic pathways present in N. meningitidis when grown on pyruvate, lactate and glucose have been investigated. NMR studies were carried out to identify how the various carbon sources were metabolized, to identify any anomalies in amino acid biosynthesis and to give information on the CAC.
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METHODS |
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Preparation of cell-free extract (CFE).
CFEs were routinely prepared from 500 ml cultures grown aerobically overnight at 37 °C, on pyruvate- or glucose-minimal medium. Cells were harvested by centrifugation (20 min, 6000 g), then resuspended in 1·0 ml 10 mM Tris/HCl, pH 8·0. Following sonication (MSE, 3x30 s, 10 µm amplitude) the debris was removed by centrifugation (10000 g, 25 min, 4 °C) and the supernatant (CFE) was stored on ice until required. CFEs for E. coli were prepared the same way, except cultures were grown overnight in BHI broth.
NMR spectroscopy.
13C- and 1H-NMR spectra were obtained on a Bruker AMX500 spectrometer operating at 125 MHz and 500 MHz for the 13C and 1H nucleus respectively. For 13C-NMR, pulses of 75° (10 µs) were used, with a spectral width of 33·3 kHz and 16 k complex points, giving an acquisition time of 0·49 s. A 4 s relaxation delay was used and the decoupler was gated on during acquisition only. Preliminary experiments and calculations indicated that these conditions permitted reasonably accurate quantitative comparisons of peak intensity, by ensuring a long enough time for relaxation of carbons with different T1 values and by not generating nuclear Overhauser effects. Spectra were Fourier transformed and integrated using FELIX 97·0 (Molecular Simulations). Samples were dissolved in D2O and run in 5 mm tubes at 35 °C. Between 1000 and 3000 scans were accumulated. 1H-NMR spectra were acquired into 8k complex points over a spectral width of 12·5 kHz and the solvent (H2O) signal was reduced by presaturation for 2 s. This is several times the T1 for the signal of interest and thus permits reliable integration of signal intensity. Samples were again run in 5 mm tubes at 35 °C. Signals in the spectra were assigned by comparison with chemical shifts stated in the literature (Rosenthal & Fendler, 1976 ; Sprott et al., 1993
; Pickett et al., 1994
). For 1H-NMR the concentration of a substrate and end product was established with reference to a standard (trimethylsilylpropionate, TSP) of known concentration, with the number of protons involved taken into account. For example, a peak for TSP and pyruvate of the same area would have concentrations in the ratio of 1:3, as the TSP peak results from nine identical protons while the pyruvate enrichment results from just three identical protons.
Preparation of samples for 1H-NMR.
Over a 1416 h time-course of growth in minimal medium [with specified carbon source(s)], samples (1·5 ml) were pelleted at intervals by centrifugation (10000 g, 25 min). The supernatant (1 ml) was removed for analysis by 1H-NMR. Growth yields were estimated after determination of the biomass protein concentration using the Lowry method, assuming that cell protein is 50% of the cell dry weight.
Fractionation of cells for 13C-NMR.
Cells were harvested (20 min, 6000 g) after overnight growth on 2- or 3-[13C]pyruvate-minimal medium (18 mM pyruvate). They were then washed in 10 mM Tris/HCl, pH 8·0, and resuspended in 1 ml 20 mM Tris/HCl buffer, pH 8·0, containing 10 mM magnesium acetate, 30 mM ammonium chloride and 6 mM mercaptoethanol. The cells were then sonicated (MSE, 3x30 s, 10 µm amplitude) and left on ice for 15 min after the addition of 0·5 mg DNase. Samples were then centrifuged at 10000 g for 30 min and the supernatant was removed. The proteins were then precipitated with ethanol (60%, v/v) at 4 °C and collected by centrifugation (10000 g, 15 min). The protein mixture was hydrolysed for 24 h in 6 M HCl, under vacuum at 110 °C, and then analysed by 13C-NMR spectroscopy.
Enzyme assays.
CFEs were prepared as described above. All assays were performed spectrophotometrically and carried out aerobically, at room temperature in 1 ml cuvettes. Changes in absorbance were followed on a Shimadzu UV-1601 dual-beam recording spectrophotometer. All enzyme assays were carried out according to Pickett et al. (1994) , with the exception of that for pyruvate kinase (Willison, 1988
). E. coli CFEs were used as the positive enzyme assay control. Protein concentration was determined using the Lowry Folin-Ciocalteau reagent.
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RESULTS |
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The predicted enrichments of key amino acids after growth on 2-[13C]pyruvate with an oxidative CAC in operation are: C-1 and C-4 for aspartate; C-1 and C-5 for glutamate and C-2 for both alanine and glycine. The predicted enrichments for growth on 3-[13C]pyruvate are: C-2 and C-3 for aspartate; C-2, C-3 and C-4 for glutamate; C-3 for alanine and no labelling for glycine (Rosenthal & Fendler, 1976 ; Sprott et al., 1993
; Pickett et al., 1994
).
Figure 1 shows a representative 13C-NMR spectrum of hydrolysed proteins after growth of cells on 2-[13C]pyruvate or 3-[13C]pyruvate. Initial inspection indicated an abundance of labelled carboxyl groups in the 2-[13C]pyruvate spectrum, while there were significantly fewer carboxyl enrichments for cells grown on 3-[13C]pyruvate. For the non-carboxyl labelling, there were substantial enrichments in the 3-[13C]pyruvate spectrum, while the labelling, in general, for these carbon atoms in the 2-[13C]pyruvate spectrum was either greatly reduced or not present. The assigned chemical shifts and the relative enrichments of individual carbon atoms of amino acids after growth on 2- and 3-[13C]pyruvate are shown in Tables 1
and 2
respectively. Individual peaks within the spectra were assigned according to previous data (Rosenthal & Fendler, 1976
; Sprott et al., 1993
; Pickett et al., 1994
).
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A more detailed examination of the derivatives of the key amino acids after growth on 2-[13C]pyruvate showed labelling consistent with the expected biosynthetic pathways. For example, threonine, isoleucine and methionine exhibited the predicted enrichments for biosynthesis from aspartate. Lysine also had predicted enrichments, although there was some additional labelling of C-3. Valine and leucine both had labelling patterns consistent with their derivation from pyruvate. Additionally, serine and cysteine, which are derived from 3-phosphoglycerate, had the predicted C-2 label, but both had an additional C-3 label.
A more comprehensive analysis of amino acid enrichments after growth on 3-[13C]pyruvate (Table 2) showed not only extensive predicted labelling, but also the presence of numerous secondary labels at lower enrichments. It was not possible to unequivocally establish the individual origins of the majority of the carboxyl labels in this case. Threonine and methionine, which are both derived from aspartate, exhibited similar labelling to their precursor. Additionally, isoleucine and lysine had three predicted enrichments. Pyruvate derivatives, valine and leucine, had several unexpected labels. Valine had an additional C-2 label, while leucine had C-1 and C-3, although these were enriched to lower levels than the expected labels. Serine and cysteine, synthesized from 3-phosphoglycerate, had a single predicted enrichment in C-3. The predicted secondary labels of aspartate, threonine and methionine were identified at the C-1 position, with the relative enrichment being very low compared to the predicted primary labels. Finally, arginine and proline had the expected labels, although arginine had an additional C-1 label enriched to a lower level than the C-2 and C-4. As mentioned previously, the presence of these secondary labels indicates a complete CAC in operation, with recycling of oxaloacetate.
Analysis of enzyme activities in N. meningitidis
The above results suggested that the N. meningitidis CAC operated in an oxidative direction when cells were grown on either 2- or 3-[13C]pyruvate. The data also implied that all the CAC enzymes were present. This was confirmed by performing individual enzyme assays using cell-free extracts of both pyruvate- and glucose-grown cells. The activities of the enzymes of the CAC are shown in Table 3.
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The oxidation of succinate to fumarate by succinate dehydrogenase was detected via a dye-linked assay. This enzyme is membrane bound and transfers electrons from succinate to enzyme-bound FAD. Electrons are subsequently channelled to the respiratory chain. Activity of succinate dehydrogenase was also identified in membrane fractions (data not shown). Two enzymes are capable of the oxidation of malate to oxaloacetate, namely malate dehydrogenase and malate:acceptor oxidoreductase or malate oxidase. Cell-free extracts of N. meningitidis did not exhibit any detectable activity of the typical NAD-dependent malate dehydrogenase. However, a dye-linked malate oxidase was detected, in CFEs and membrane fractions (data not shown). This enzyme is thought to be FAD dependent (Holten, 1976b ) and is probably the malate:quinone oxidoreductase (NMB2069) predicted in the genome sequence (Tettelin et al., 2000
).
Anaplerotic CO2-fixation enzymes, including PEP carboxylase, PEP carboxykinase and pyruvate carboxylase, were assayed. Of these, only PEP carboxylase was detectable (Table 3). The ATP-dependent synthesis of PEP from pyruvate can be achieved via two enzymes, PEP synthetase and pyruvate phosphate dikinase (Cooper & Kornberg, 1974
). A low PEP synthetase activity was detected in glucose-grown cells but not in pyruvate-grown cells. Pyruvate kinase was detected at a similar specific activity in both pyruvate- and glucose-grown cells.
Extremely high activity corresponding to phosphotransacetylase (PTA) was detected in CFEs (Table 4). Acetate kinase (ACK), the subsequent enzyme in the conversion of acetyl-CoA to acetate, also had detectable activity, although considerably lower than that of phosphotransacetylase (Table 4
). To confirm the presence of ACK, P1,P5-di(adenosine-5') pentaphosphate (AP5A), which inhibits acetate thiokinase but not acetate kinase activity (Lienhard & Secemski, 1973
), was used. It was demonstrated that the reaction was dependent on acetate but not CoA and was not inhibited by AP5A (data not shown).
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1H-NMR analysis of substrate utilization and acetate release during growth on various carbon sources
To study patterns of carbon utilization and end product excretion, 1H-NMR analysis was carried out. Preliminary experiments involving growth of N. meningitidis on pyruvate showed that by the end of the growth period a significant amount of acetate was excreted into the medium. Acetate excretion occurred even though the bacteria were grown aerobically in minimal medium where most of the pyruvate present would be expected to enter the CAC and be oxidized to CO2.
More detailed investigations of the utilization of the carbon source and the release of the end product were carried out in order to establish the patterns of utilization and excretion, and the relationship between carbon source and acetate production (Table 5). Growth on pyruvate led to 42% of the initial pyruvate being converted to acetate and excreted by the end of the experiment. In contrast, when lactate was provided as the sole carbon source, only 13% of the substrate was converted to acetate. The growth yield from pyruvate was 40 g mol-1, while for DL-lactate the growth yield was only 19 g mol-1. When glucose was supplied as a sole carbon source 34% of the glucose was converted to acetate (growth yield of 105 g mol-1).
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DISCUSSION |
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The direct comparison of labelling of key amino acids derived from cells grown on 2-[13C]pyruvate and 3-[13C]pyruvate (Fig. 1) allowed the overall nature of the CAC in N. meningitidis to be assessed. The abundance of carboxyl labels in amino acids after growth on 2-[13C]pyruvate was an initial indicator that an oxidative CAC was operating. Additional evidence included the labelling of the central carbons of glutamate and aspartate (C-2, C-3 and C-4, and C-2 and C-3, respectively) in the spectra from cells grown on 3-[13C]pyruvate. Also, the presence of the C-2 alanine and glycine labels, but the absence of the C-3 alanine label (2-[13C]pyruvate growth), was indicative. The identification of numerous secondary labels in a variety of amino acids, from growth on both 2- and 3-[13C]pyruvate, is also indicative that the CAC is complete.
A number of peaks in the spectra could not be assigned unambiguously. This is particularly evident for the carboxyl groups, where there are a large number of enrichments with similar chemical shifts. Hence there are several predicted carboxyl labels in several amino acids that could not be identified, for example the expected threonine C-1 (3-13C), glutamate C-5 (2-13C) and lysine C-1 (3-13C) were not apparent. Secondly there was a certain extent of scrambling, which leads to unexpected enrichments, such as the C3 from methionine, leucine, glutamate, lysine, serine and cysteine (all from growth on 2-[13C]pyruvate). Scrambling arises due to the bacteria recycling and degrading amino acids and CAC intermediates.
The presence of a fully functional CAC enzyme complement was established in this study, supporting the data obtained in the 13C-NMR. It should be noted that the meningococcal genome sequence contains two genes for both aconitase and fumarase (Tettelin et al., 2000 ); therefore the activity we have observed may not be due to a single enzyme in these cases. PEP synthetase and pyruvate kinase (PK) were identified in the meningococcal genome sequence (Tettelin et al., 2000
) and were detected via conventional enzyme assays in this study. However, PEP synthetase could not be detected in pyruvate-grown cells. The coupled assay used may not be suitable for the detection of low activities of this enzyme. PK is the final step in glycolysis, converting PEP to pyruvate, but the specific activities of this enzyme were similar in both pyruvate-grown cells and glucose-grown cells.
An extremely high PTA activity was demonstrated in this study, although ACK had a much lower activity. In most bacteria, these enzymes form part of a fermentative pathway, with acetate as the end product. However, PTA appears to be a key enzyme in meningococcal metabolism during growth on glucose, pyruvate, or DL-lactate. It utilizes the pivotal metabolic intermediate acetyl-CoA to produce acetyl phosphate. The main effect of this is to reduce the level of energy conservation from an individual carbon source because of the conversion of carbon to acetate production and not through oxidation via the CAC.
From 1H-NMR data, it was apparent that pyruvate, glucose and lactate were metabolized by both the CAC and the PTAACK pathways, the latter resulting in the excretion of acetate. This is in agreement with previous investigations in the closely related bacterium N. gonorrhoeae (Hebeler & Morse, 1976 ). When meningococci were grown on either pyruvate, glucose, or lactate, it was found that they exhibited very different levels of acetate production and growth yield. As would be expected, growth on glucose gave a higher growth yield than for pyruvate or lactate. Apart from extra ATP produced by substrate-level phosphorylation during glucose catabolism to pyruvate, both lactate and pyruvate need to feed into the gluconeogenesis pathway as well as the CAC in order to allow synthesis of carbohydrates, resulting in lower growth yields compared to growth on a sugar. Growth on DL-lactate gave both the lowest growth yield and the least acetate excretion. This contradicts the work of Erwin & Gotschlich (1993)
, who showed that N. meningitidis was able to grow on lactate at least as well as on glucose. Growth on lactate requires the presence of LDH; Erwin & Gotschlich (1996
) demonstrated the presence of at least two L-lactate-specific LDHs and one D-lactate-specific LDH in N. meningitidis. This has recently been confirmed by the genome sequence (Tettelin et al., 2000
). The bioenergetics of growth on lactate by N. meningitidis is clearly less favourable than growth on pyruvate. This is possibly due to the utilization of NAD, but N. meningitidis possesses a cytoplasmic LDH, the activity of which is greater with NADH, which favours the formation of lactate from pyruvate (Holten & Jyssum, 1974
). Therefore, the oxidation of lactate to pyruvate is thought to occur via two membrane-associated LDHs that are specific for either L- or D-lactate and feed electrons directly from lactate into the electron-transport chain at the level of the quinone pool (Erwin & Gotschlich, 1993
, 1996
). Thus, growth on lactate could result in over-reduction of the quinone pool and lead to redox-balancing problems for the bacteria, which may lower the growth yield compared to growth on pyruvate.
Large amounts of acetate were shown to be excreted into the medium during growth of N. meningitidis on pyruvate. This has been found in other bacteria during growth where the carbon source is in excess and may represent an overflow metabolism due to the inability to control carbon-substrate uptake (Elmansi & Holms, 1989 ). The consumption of carbon is in excess of that needed to support the growth rate and the excess carbon is excreted as acetate.
The analysis of PTA and ACK activities, on a range of carbon sources, was carried out in order to establish whether there was a relationship between the amount of acetate excreted into the medium and the relative PTA and ACK specific activities. When meningococci were grown on a single carbon source there appeared to be a pattern of related activity between PTA and ACK, suggesting co-regulation. During growth on a single carbon source the highest PTA activity also corresponded to the highest ACK activity. However, growth on dual carbon sources resulted in an inverse relationship, where the lowest PTA activity corresponded to the highest ACK activity. In other prokaryotes the genes for both of these enzymes are usually co-transcribed in a single operon (Boynton et al., 1996 ; Latimer & Ferry, 1993
; Matsuyama et al., 1994
; Summers et al., 1999
). However, the genome sequence of N. meningitidis has, unusually, revealed the presence of two separate ACK genes which are unlinked to the gene encoding PTA (Tettelin et al., 2000
). This arrangement may explain the variation in the PTA and ACK activities observed in this study.
Interestingly, the product of the PTA reaction, acetyl phosphate, has been implicated as a global regulator (Nyström, 1994 ; Wanner, 1992
; McCleary et al., 1993
; McCleary & Stock, 1994
; Wanner & Wilmes-Reisenberg, 1992
). This is thought to occur via the replacement of the phosphate donor in two-component regulatory systems, where acetyl phosphate acts directly on the response regulator (Wanner, 1992
). However, the physiological significance of this remains controversial (McCleary, 1996
). Growth on different carbon sources located at diverse sites within the human host will affect both PTA and ACK activities and alter the intracellular pool of acetyl phosphate. This could possibly be used as a global signal by the bacterium during pathogenesis. Indeed, mutants of PTA and pyruvate oxidase, another enzyme that converts pyruvate to acetyl phosphate, affect the virulence of Vibrio cholerae and Streptococcus pneumoniae, respectively (Chiang & Mekalanos, 1998
; Spellerberg et al., 1996
).
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ACKNOWLEDGEMENTS |
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Received 11 December 2000;
accepted 14 February 2001.