Comparison of gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes

Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol ¹³C enrichment after infusion of labeled precursor ([¹³C_1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.     

13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry

¹³C-based metabolic flux analysis (¹³CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured ¹³C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-¹³C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-¹³C] glucose. Decreasing the [1-¹³C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected ¹³C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

A three-phase liquid chromatographic method for δC analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ¹³C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ¹³C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ¹³C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ¹³C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.     

Novel three-dimensional 1H−13C−31P triple resonance experiments for sequential backbone correlations in nucleic acids

Summary Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, ¹H–¹³C–³¹P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of ¹H–³¹P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihydral angle constraints. By relying on ¹H–³¹P rather than ¹³C–³¹P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of ¹H–³¹P and ¹³C–³¹P couplings provide dihedral angle constraints for RNA structure determination.     

Metabolic network analysis on Phaffia rhodozyma yeast using C–labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for ‘all natural’ products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-¹³C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes.Amino acid labeling patterns, as determined by GC–MS, were in accordance with a metabolic network consisting of the Embden–Meyerhof–Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Analysis of tert.-butyldimethylsilyl [1-C]palmitic acid in stool samples by gas chromatography–mass spectrometry with electron impact ionisation: comparison with combustion isotope-ratio mass spectrometry

The use of ¹³C-labelled compounds to study lipid metabolism is increasing. Typically less than 40% of the orally administered label is recovered in breath CO₂. The remainder must be either absorbed and not oxidised or not absorbed and remain in the faeces. Two methods of determining how much tracer passes through the body, and is present in the stool, were compared. Compound specific analysis of tert.-butyldimethylsilyl [¹³C]hexadecanoic acid by gas chromatography–mass spectrometry (GC–MS) with electron impact ionisation was compared with bulk analysis of whole stool and lipid extract by continuous flow isotope ratio mass spectrometry (CF–IRMS) with a combustion interface. The mean difference between the IRMS and GC–MS methods was −0.02 mmol ¹³C d⁻¹ with a mean excretion of 14.2 mmol ¹³C d⁻¹. Combustion IRMS is both simpler and cheaper, when the objective is to determine how much administered dose appears in stool, and information about the form of the label is not required.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

13CMFA过程中GC-MS分析菌体蛋白氨基酸的13C标记丰度

基于13C标记的代谢通量分析(13CMFA)由于具有准确性和应用性已成为国际上代谢工程研究的热点,其中一个至关重要的问题就是分析菌体蛋白氨基酸的13C标记丰度信息。用含20%全标记葡萄糖（［U-13C］）和80%天然葡萄糖的合成培养基喂养维生素B12生产菌Pseudomonas denitrifican,然后在稳态条件下取20mg带13C标记菌体用1ml 6mol/L盐酸95℃水解24h得到带13C标记的菌体蛋白氨基酸;氨基酸经分离、浓缩、真空干燥、MBDSTFA衍生化后得到的TBDMS衍生物可以用气相色谱-质谱(GC-MS)进行分析,最后分析质谱图得到15种菌体蛋白氨基酸的13C标记丰度信息。成功获得菌体蛋白氨基酸13C标记丰度信息的实验方法和样品处理技术,为13CMFA在国内进一步发展提供了参考意义。     

C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY)

¹³C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of ¹³C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a ¹³C NMR spectrum. We demonstrate here that groups of glutamate ¹³C isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the ¹³C fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct ¹³C NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of ¹³C isotopomer analysis to tissue samples not amenable to direct ¹³C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

Impact of Agrobacterium tumefaciens-induced stem tumors on NO3− uptake in Ricinus communis

Developing tumors induced by Agrobacterium tumefaciens, strain C58, on stems of Ricinus communis L. var. gibsonii cv. Carmencita were shown to be strong metabolic sinks for sucrose and amino acids, thus causing higher nutrient demand in the host plant. However, NO₃ ^– uptake and, to a lesser extent, also H₂PO₄ ^– uptake were strongly inhibited. Correspondingly, NO₃ ^– concentration was lower in tumorised than in the control plants. NO₃ ^–reductase activity was the same in both plant types, but it was completely suppressed in the tumors. The electrical membrane potential difference of root cells was unaffected in tumorised plants when soil-grown, but significantly lowered when grown hydroponically. Consistent with the low NO₃ ^– uptake rate, NO₃ ^–-dependent membrane depolarisation at the onset of NO₃ ^–/2H⁺-cotransport was nearly zero. In the phloem sap, sucrose and amino acid concentrations were considerably lower in tumorised than in control plants, and lower below than above the tumor. The qualitative pattern of amino acids of the phloem sap of stems was almost the same in tumorised and control plants. It is concluded that neither the overall amino acid concentration nor special amino acids nor ammonium in the transport phloem suppress NO₃ ^– uptake in the roots. Aminocyclopropane-carboxylate, the precursor of ethylene, which is produced in the tumors in high amounts, was low in the stems and the same in both plant types. Thus, ACC and ethylene were ruled out as directly interfering with nutrient uptake in the roots. Root morphology was strongly affected during tumor development. Root fresh weight decreased to 50% of the controls and lateral root development was almost completely prevented. This suggests that the high tumor ethylene production, together with an increasing concentration of phenolic compounds, severely inhibits the basipetal auxin flow to the roots. Auxin accumulation and retention was confirmed by specifically enhanced expression of the auxin-responsive promoter of the soybean gene GH3:GUS in tumors induced in transgenic Trifolium repens L. Hence, root development is poorer and anion uptake inhibited in tumorised plants. This may be aggravated by abscisic acid accumulation in the tumor and its basipetal export into the roots. Moreover, sucrose depletion of the sieve tubes leads to energy shortage at the root level for maintaining energy-dependent anion uptake.     

Identification of hexose hydrolysis products in metabolic flux analytes: a case study of levulinic acid in plant protein hydrolysate

Biosynthetically directed fractional ¹³C labeling, a popular methodology of metabolic flux analysis, involves culture on a mixture of ¹³C and ¹²C substrates and preparation a ‘metabolic flux analyte’ (typically protein hydrolysate) from the biomass. Metabolic flux analytes prepared from complex eukaryotes may contain additional compounds than those prepared from microorganisms. We report the presence of such compounds (hexose hydrolysis products) in a plant metabolic flux analyte (acid hydrolyzed protein from soybean embryos). We designed NMR experiments to systematically identify these compounds, and found that they were levulinic acid (LVA; major) and hydroxyacetone (HyA; minor). These acid hydrolysis products of hexoses (glucose and mannose) were generated during acid hydrolysis of glycosylating sugars (glucosamine and mannose) associated with soybean embryo protein. Analysis of LVA by two-dimensional [¹³C, ¹H] NMR and measurement of its J-coupling constants revealed long-range coupling between atoms C3 and C5, which enables LVA to provide more isotopomer information than its precursor hexose. Furthermore, we found that LVA and HyA preserve the isotopomeric composition of the metabolic hexose from which they are derived. An important consequence of these results is that comparison of LVA and HyA isotopomers from two separate metabolic flux analytes (protein hydrolysate and starch hydrolysate) from the same plant tissue can distinguish between parallel glycolysis and pentose phosphate pathways in different subcellular compartments.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Amino acids as a nitrogen source for tomato seedlings: The use of dual-labeled (C, N) glycine to test for direct uptake by tomato seedlings

Direct uptake of organic nitrogen (ON) compounds, rather than inorganic N, by plant roots has been hypothesized to constitute a significant pathway for plant nutrition. The aim of this study was to test whether tomatoes (Solanum lycopersicum cv. Huying932) can take up ON directly from the soil by using ¹⁵NH₄Cl, K¹⁵NO₃, 1, 2-¹³C₂¹⁵N-glycine labeling techniques. The ¹³C and ¹⁵N in the plants increased significantly indicating that a portion of the glycine-N was taken up in the form of intact amino acids by the tomatoes within 48 h after injection into the soil. Regression analysis of excess ¹³C against excess ¹⁵N showed that approximately 21% of the supplied glycine-N was taken up intact by the tomatoes. Atom% excesses of ¹⁵N and ¹³C in the roots were higher than in any shoots. Results also indicated rapid turnover of amino acids (e.g., glycine) by soil microorganisms, and the poor competitive ability of tomatoes in absorbing amino acids from the soil solution. This implies that tomatoes can take up ON in an intact form from the soil despite the rapid turnover of organic N usually found under such conditions. Given the influence of climatic change and N pollution, further studies investigating the functional ecological implications of ON in horticultural ecosystems are warranted.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

Quantitative isotopic C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin

Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in ¹³C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ–NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ–NMR methodology is sufficiently precise and robust to measure the isotope shifts in the ¹³C/¹²C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl–CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl–CoA as intermediate in the catalytic pathway.     

13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.