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Background The pstSCAB operon of Corynebacterium glutamicum, which encodes an ABC transport system for uptake of phosphate (Pi), is induced during the Pi starvation response. The two-component regulatory system PhoRS is involved in this response, but partial Pi starvation induction of pstSCAB in a ΔphoRS mutant indicated the involvement of additional regulator(s). Regulation of pstSCAB also involves the global transcriptional regulator GlxR. Results DNA affinity chromatography identified the regulator of acetate metabolism RamB as a protein binding to pstS promoter DNA in vitro. Gel mobility shift assays and mutational analysis of the pstS promoter region revealed that RamB binds to two sites localized at positions −74 to −88 and −9 to +2 with respect to the transcriptional start site of pstSCAB. Reporter gene studies supported the in vivo relevance of both binding sites for activation of pstSCAB by RamB. DNA microarray analysis revealed that expression of many Pi starvation genes reached higher levels during the Pi starvation response on minimal medium with glucose as sole carbon source than in Pi starved acetate-grown C. glutamicum cells. Conclusions In C. glutamicum, RamB is involved in expression control of pstSCAB operon. Thus, transcriptional regulation of pstSCAB is complex involving activation by the phosphate-responsive two-component regulatory system PhoSR and the regulators of carbon metabolism GlxR and RamB.

Physiological, biochemical and genetic studies of Corynebacterium glutamicum, a workhorse of white biotechnology used for amino acid production, led to a waste knowledge mainly about amino acid biosynthetic pathways and the central carbon metabolism of this bacterium. Spurred by the availability of the genome sequence and of genome-based experimental methods such as DNA microarray analysis, research on genetic regulation came into focus. Recent progress on mechanisms of genetic regulation of the carbon, nitrogen, sulfur and phosphorus metabolism in C glutamicum will be discussed.

Positron emission tomography (PET) and nuclear magnetic resonance spectroscopy (MRS) are two biomedical measurement techniques developed in the end of the XXth century, which drastically improved the amount of accessible information available in vivo. PET became popular through the most widely used tracer, fluorodeoxyglucose (FDG), which enables the measurement of the local glucose utilization and is nowadays routinely applied in clinical practice. Nuclear magnetic resonance is used in a large array of applications such as in analytical chemistry or chemical structure determination and is essentially known for its versatile medical imaging capabilities, grouped under the name of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). In vivo MRS measures concentrations of metabolites by interacting with various nuclei such as 1H, 13C or 15N, with the major asset that MRS enables the identification of the chemical position at which the detected nuclei is located in the measured molecule. When coupled with labeled substrate infusions, dynamic MRS gives the opportunity to probe specific biochemical reactions directly in vivo, which opens the way to a wide range of metabolic studies. However, the correct interpretation of the elaborate dynamic labeling data acquired either with MRS or PET tracer experiments requires the application of adapted metabolic models to derive quantitative metabolic rates characterizing the biochemical processes under study. The work of this thesis involves both PET and MRS studies of brain energy metabolism in rodents and focuses on the development of adapted metabolic models to derive reliable metabolic fluxes characterizing various brain metabolic processes, such as glucose consumption, glial and neuronal oxidative metabolism and neurotransmission. FDG-PET enables the measurement of the cerebral metabolic rate of glucose (CMRGlc) using a three-compartment metabolic model for FDG transport across the blood-brain barrier and FDG phosphorylation to FDG-6P. Although this method is well established, a main drawback of CMRGlc measurement with FDG-PET is the necessity to characterize the available FDG concentration in the plasma over the experiment duration, the so-called arterial input function. This constraint is a strong limitation for preclinical studies in rodents, due to their low blood volume. We developed therefore a new method to extract the FDG input function directly from the PET images in rats and mice, using the time-activity curve of a voxel located in the inferior vena cava. The method was validated by comparison with a dedicated external blood counter and manual blood sampling. Using this method, a CMRGlc of ~0.22 μmol/g/min was determined in the mouse cortex. Glial oxidative metabolism can be specifically assessed using glial specific substrates, in particular [1-11C] acetate. However, no existing metabolic model described the 11C tissue activity curve in terms of neuroglial energy metabolism. In order to extract quantitative metabolic fluxes characterizing the glial TCA cycle rate and glutamate/glutamine cycling, we adapted the neuroglial two-compartment modeling approach previously used in 13C MRS cerebral metabolic studies to interpret positron emission data following 11C-acetate injection in the rat. The precision and accuracy of the estimated metabolic parameters was tested and a composite glial metabolic flux Vgtg=0.136 ± 0.042 μmol/g/min and a neurotransmission flux Vnt=0.170 ± 0.103 μmol/g/min were obtained. This approach enabled a direct comparison of the metabolic fluxes measured using 11C PET with the values determined with 13C MRS. In the field of 13C MRS, we took advantage of the higher sensitivity and spectral resolution available at high magnetic field (14.1T) to measure glial and neuronal oxidative metabolism, glutamatergic neurotransmission and pyruvate carboxylation in more details and with higher precision using both [1,6-13C2] glucose and [2-13C] acetate infusions in rats. In the acetate study, the glial specific uptake of acetate enabled a quantification of glial oxidative metabolism with unrivalled precision. We could estimate for the first time separately the transmitochondrial flux Vx in the glial and neuronal compartment, a biochemical pathway which has been subject of strong controversy. The glial and neuronal Vx were determined with high precision, as calculated through Monte Carlo simulation, and their value was on the same order of magnitude than the respective glial and neuronal TCA cycle flux. The model was extended to estimate the fraction of glutamate located in the glial compartment. A glial glutamate concentration of 0.6 ± 0.1 μmol/g was found. The values of the different metabolic fluxes characterizing the neuroglial system (Vtcag=0.27, Vxg=0.17, VPC=0.087, VNT=0.15, Vtcan=0.37 and Vxn=0.46 μmol/g/min) were in very good agreement with the values found using [1,6-13C2] glucose infusion. In addition, the effect of the MRS temporal resolution, experiment duration and signal- to-noise ratio on the precision of the derived metabolic fluxes was analyzed. Previous simulation studies showed that two-compartment modeling of neuroglial metabolism using 13C glucose infusion could lead to unreliable parameter estimations. However, using polarization transfer 13C MRS at 14.1T, the carbon positions C4, C3 and C2 of glutamate and glutamine as well as the positions C3 and C2 of aspartate could be measured with high SNR. The C2 positions were used in two-compartment modeling for the first time, enabling a precise measurement of the apparent glutamatergic neurotransmission and glial metabolism, including pyruvate carboxylation, as confirmed by Monte Carlo simulation. The glial dilution at the level of acetyl-CoA, which is related to the metabolism of other substrates than glucose, could also be characterized. Brain metabolism under hyperammonemic conditions was probed with 15N MRS under labeled ammonia infusion and modeled using a modified neuroglial two- compartment model describing the dynamics of [5-15N] glutamine, [2-15N] glutamate and [2-15N] glutamine. Due to detoxification processes, the total glutamine concentration, measured with 1H MRS, was steadily increasing. This non-steady- state metabolic condition resulting from the hyperammonemic stress was taken into account in the model, resulting in the determination of the apparent neurotransmission (0.26 ± 0.030 μmol/g/min), the glutamate dehydrogenase (0.029 ± 0.002 μmol/g/min) and net glutamine accumulation (0.033 ± 0.001 μmol/g/min). Finally, compartmental modeling was applied to 13C labeling studies in the awake rat, fed during 5, 24 or 48 h with a [1-13C]-labeled glucose solution, to determine brain glycogen and NAA turnover times (τGlyc=5.3 ± 3.2 h and τNAA=15.6 ± 6.5 h), using 13C spectra of brain extracts. A group of rats followed a mild brain activation protocol over 5 hours, which resulted in a decreased glycogen turnover time (τGlyc =2.9 ± 1.2 h).

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

Thomas Graham, a Scotsman who lived from 1805–1869, demonstrated that vegetable parchment acts as a semipermeable membrane1. He coated parchment with albumin to close any defects then stretched it over a wooden or guttapercha hoop which he floated in a tank of water. Into the hoop he put a combination of crystalloid and colloid and demonstrated that only the crystalloid diffused through the parchment into the water. Graham coined the term dialysis for this phenomenon. He later repeated the experiment using urine and again showed that only the crystalloid matter passed into the water, and that when the water was evaporated to dryness it yielded a white crystalloid powder which was mainly urea. Graham was a chemist rather than a physician and as such did not apply dialysis to animals.

DURING studies on the acetate metabolism of Penicillium chrysogenum in this Department, it was found that alcoholic extracts of acetate-grown mycelium contained considerable quantities of glutamic acid and glycine, as well as a number of other metabolites1.

To evaluate the acid-base status and plasma acetate levels of children undergoing hemodialysis, we studied 8 children during 11 hemodialysis sessions. During dialysis, the blood bicarbonate fell (20.5±0.7 to 19.6±0.8 mEq/L), the pCO2 fell (33.4±0.8 to 27.5±1.4 mm Hg), and the pH rose (7.42±0.01 to 7.48±0.02). During the hour after dialysis the bicarbonate rose to normal, 23.4±9.7 mEq/L, the pC02 rose to 32.8±0.8 mm Hg, and the pH remained unchanged. The half-life of plasma acetate, measured after dialysis, was 8.7±0.9 minutes. During 5 “high efficiency” dialysis sessions (urea clearance>3.0 ml/min/kg) blood bicarbonate fell 3.2 mEq/L, pCO2 fell 8.7 mm Hg, and plasma acetate rose to 7.51 mM/L, whereas during 6 “routine efficiency” dialysis sessions (urea clearance 1.5 - 3.0 ml/min/kg) bicarbonate rose 1.0 mEq/L, pCO2 fell 3.7 mm Hg, and acetate rose to 3.52 mM/L. At one hour after the end of dialysis blood bicarbonate, pCO2, and plasma acetate concentrations were similar in the two groups. Clinical problems occurred more frequently in the high efficiency group during dialysis but the difference was not significant. The data indicate that 1) dialysis with acetate buffer effectively corrects pre-dialysis metabolic acidosis, 2) although children have a high rate of acetate metabolism, during high efficiency dialysis this rate is exceeded by the influx of acetate, and acid-base abnormalities occur. These abnormalities are transient but may cause clinical problems.

Total entry rate of blood glucose and the rate of irreversible loss of blood acetate and its oxidation have been examined in sheep at rest and while walking on a horizontal treadmill at 5 km/h for 2 h. Sheep were given their daily ration of 1000 g chaff in 24 eaual portions at hourly intervals and received multiple intravenous injections of [2-3H]glucose and intravenous infusions of [1-14C]acetate and NaH14CO3. At rest the total entry rate of blood glucose was 0-44 +/- 0-03 mmol/min (values given as mean +/- s.e.m. for four sheep), the glucose pool was 23 +/- 1 mmol and the rate of irreversible loss of blood acetate was 2-3 +/- 0-1 mmol/min. During exercise, the total entry rate of blood glucose was 0-84 +/- 0-04 mmol/min, the glucose pool was 27 +/- 2 mmol and the rate of irreversible loss of blood acetate was 2-6 +/- 0-1 mmol/min. Gluconeogenesis apparently increased markedly in response to exercise as indicated by the incorporation of 14C from blood bicarbonate into blood glucose. Despite the substantial increase in the rate of irreversible loss of blood bicarbonate (from 11-6 +/- 1 to 20-2 +/- 2 mmol C/min), and hence energy expenditure with exercise, only a slight change was recorded in the proportion of the irreversible loss rate of acetate that was oxidized.

Euglena gracilis, a flagellate protozoan, utilizes acetate and ethanol as energy and carbon sources. The acetate metabolism of Euglena grown on ethanol differs from that grown on acetate. Ethanol-grown cells have a rate of acetate oxidation at pH 7.0 lower than that of acetate-grown cells1. However, ethanol-grown cells incubated in the presence of acetate for about 80 min. often adapt to acetate, taking on characteristics similar to those of acetate-grown cells1,2. Thus, it was considered of interest to determine the respiration of Euglena in the presence of both acetate and ethanol.