The assimilation of carbon by Chloropseudomonas ethylicum

1. The enzymes in ultrasonically prepared extracts of Chloropseudomonas ethylicum were studied to elucidate how this organism assimilates acetate and carbon dioxide and why it cannot grow with either of these two compounds alone. 2. Such extracts can (i) convert acetate and oxaloacetate into alpha-oxoglutarate, (ii) convert oxaloacetate into succinyl-CoA, (iii) convert phosphopyruvate into 3-phosphoglyceraldehyde and (iv) interconvert phosphopyruvate and pyruvate via oxaloacetate. 3. Pyruvate kinase, alpha-oxoglutarate dehydrogenase, ribulose diphosphate carboxylase, isocitrate lyase and malate synthase were not detected. 4. It is difficult to detect aconitate hydratase, fumarate hydratase and citrate synthase in extracts of the organism ultrasonically treated in tris buffer; to demonstrate these enzymes extracts should be prepared in phosphate buffer containing 2-mercaptoethanol. 5. Provided that this organism can synthesize pyruvate from acetate and carbon dioxide, the enzymes detected are sufficient to account for the nutritional requirements of this organism.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Fermentation of trans-aconitate via citrate,oxaloacetate, and pyruvate by Acidaminococcus fermentans

Growing cells of Acidaminococcus fermentans (DSM 20731 and ATCC 25085) fermented trans-aconitate via citrate, oxaloacetate, and pyruvate to approximately 2 CO₂, 1.8 acetate, 0.1 butyrate and 0.9 H₂. The carbon and electron recoveries were close to 100%. On citrate no growth was observed and washed cells were unable to ferment this tricarboxylate. In cell-free extracts, however, citrate as well as trans-aconitate were readily fermented to CO₂ and acetate. Under these conditions, also cis-aconitate, oxaloacetate, and pyruvate were formed, whereas butyrate and intermediates of glutamate fermentation, 2-oxoglutatrate and glutaconate, could not be detected. Citrate Si-lyase, a Mg²⁺-dependent oxaloacetate decarboxylase, and pyruvate synthase were present in quantities that corresponded to the growth rate of the organism. Received: 3 May 1996 / Accepted: 12 August     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Pathway of Succinate and Propionate Formation in Bacteroides fragilis

Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with (14)C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and propionyl-coenzyme A. From the results it also is clear that CO(2) is necessary for growth because it is needed for the formation of C4 acids. There was also a randomization of carbons 1, 2, and 6 of glucose during conversion to acetate, which indicated that pyruvate kinase played a minor role in pyruvate formation from phosphoenolpyruvate. Phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase, and malic enzyme (nicotinamide adenine dinucleotide phosphate-dependent) were present in cell extracts of B. fragilis, and the results of the labeling experiments agreed with pyruvate synthesis via oxaloacetate and malate if these acids are in equilibrium with fumarate. The conversion of [2-(14)C]- and [3-(14)C]lactate to acetate was not associated with a randomization of radioactivity.     

The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

Metabolic pathways for cytotoxic end product formation from glutamate- and aspartate-containing peptides by Porphyromonas gingivalis

Metabolic pathways involved in the formation of cytotoxic end products by Porphyromonas gingivalis were studied. The washed cells of P. gingivalis ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of P. gingivalis. P. gingivalis cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.     

A direct pathway for the conversion of propionate into pyruvate in Moraxella lwoffi

1. The identity of the organism previously known as Vibrio O1 (N.C.I.B. 8250) with a species of Moraxella is established. 2. The ability of cells to oxidize propionate is present only in cells with an endogenous respiration and this ability is increased 80-fold when the organism is grown with propionate. 3. Isocitrate lyase activity in extracts from propionate-grown cells is the same as that in extracts from lactate-grown cells, about tenfold greater than that in extracts from succinate-grown cells and slightly greater than half the activity in extracts from acetate-grown cells. 4. With arsenite as an inhibitor conditions were found in which the organism would catalyse the quantitative oxidation of propionate to pyruvate. When propionate was completely utilized pyruvate was metabolized further to 2-oxoglutarate. 5. The oxidation of propionate by cells was incomplete both in a ;closed system' with alkali to trap respiratory carbon dioxide and in an ;open system' with an atmosphere of oxygen+carbon dioxide (95:5). Acetate accumulated. Under these conditions [2-(14)C]- and [3-(14)C]-propionate gave rise to [(14)C]acetate. The rate of conversion of [2-(14)C]propionate into (14)CO(2), although much less than the rate of conversion of [1-(14)C]propionate into (14)CO(2), was slightly greater than the rate of conversion of [3-(14)C]propionate into (14)CO(2). 6. The oxidation of propionate by cells was complete in an ;open system' with an atmosphere of either oxygen or air. Under these conditions very little [1-(14)C]propionate was converted into (14)C-labelled cell material. The conversion of [2-(14)C]- and [3-(14)C]-propionate into (14)C-labelled cell material occurred at an appreciable rate, the rate for the incorporation of [3-(14)C]propionate being slightly more rapid. In the absence of a utilizable nitrogen source part of the [(14)C]propionate was incorporated into some reserve material, which was oxidized when added substrate had been completely utilized. 7. [(14)C]-Pyruvate produced from [(14)C]propionate was chemically degraded. The C((1)) of propionate was found only in C((1)) of pyruvate. At least 86% of C((2)) of pyruvate was derived from C((2)) of propionate and at least 92% of C((3)) of pyruvate from C((3)) of propionate. 8. These results are incompatible with the operation of any of the previously described pathways for propionate metabolism except the direct one, perhaps via an activated acrylate.     

A degradation pathway of propionate in Salmonella typhimurium LT-2

Salmonella typhimurium LT-2 can utilize propionate as its sole carbon source. Studies on growth, oxidation by resting cell suspensions and by permeabilized cells, suggest that the propionate is transported by the acetate system. This result was confirmed using labeled propionate and acetate. ATP-monocarboxylate phosphotransferase, acyl-CoA orthophosphate acyl-transferase, propionyl-CoA dehydrogenase, acrylyl-CoA hydratase, lactate dehydrogenase, phosphoenolpyruvate (PEP) synthase and PEP-carboxylase activities have been identified in extracts of cells grown on propionate. Mutants deficient in PEP-carboxylase and synthase are unable to utilize propionate. On the basis of results obtained, it seems that the propionate degradation pathway occurs via acrylate and that PEP-synthase and PEP-carboxylase are essential enzymes.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.     

Pyruvate metabolism by a nitrogen-fixing bacterium

1. The major products of pyruvate dissimilation by washed intact cells of Achromobacter N4-B under nitrogen-fixing conditions are acetate and formate. The formation of succinate and isocitrate and the assimilated amino acids requires carbon dioxide fixation. 2. The products formed by cells incubated with pyruvate in an atmosphere of nitrogen were compared with those formed by cells incubated in an atmosphere of helium. Production of hydrogen and the formation of succinate were greater under helium than under nitrogen. Production of acetate and formate and the utilization of pyruvate were the same in both atmospheres. 3. Cell-free preparations, unlike intact cells of Achromobacter N4-B, do not evolve hydrogen, but do produce lactate. 4. It is suggested that, in cell-free preparations incapable of fixing nitrogen, electrons are accepted from pyruvate to form lactate rather than being used for the reductive formation of ammonia and hydrogen.     

Energy conservation by succinate decarboxylation in Veillonella parvula.

Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4-3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA:pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

Enzymic studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

Cell-free extracts of Peptostreptococcus elsdenii, a strict anaerobe from the rumen, were examined for enzymes catalysing the steps in the biosynthesis from lactate of alanine, serine, aspartate and glutamate. Extracts contain the enzymes necessary for the formation of alanine from lactate via pyruvate. The presence of enzymes catalysing the interconversion of phosphoglycerate and phosphohydroxypyruvate, the transamination of the latter to phosphoserine and the cleavage of phosphoserine to serine and inorganic phosphate was demonstrated, suggesting that serine is formed via these intermediates. ;Malic' enzyme, malate dehydrogenase and glutamate-oxaloacetate transaminase are present in extracts and could account for aspartate formation. The extracts catalyse all of the steps of the tricarboxylic acid pathway leading from oxaloacetate plus acetate to glutamate. Together with substantive data from previous radioactive tracer studies the results provide strong evidence that these four amino acids are synthesized in this strict anaerobe by pathways closely similar to those operating in aerobic and facultatively aerobic organisms.     

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.