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Biomedical subjects

O M Neijssel

Publications and source records attributed to O M Neijssel.

At least 19 recordsLinked to original sources
PubMed1997

Bioenergetic consequences of microbial adaptation to low-nutrient environments.

A striking property of many prokaryotes is their enormous metabolic flexibility with respect not only to catabolic and anabolic substrates but also with respect to the continuously changing availability of nutrients. The phenotypic responses to low-nutrient growth conditions involve structural changes in the cellular make-up, changes in the specific capacity of the enzyme system(s) involved in uptake and/or assimilation of the limiting nutrient and changes in the affinity of these enzymes. Here the responses of some members of the Enterobacteriaceae to potassium-, ammonium- and energy source-limited conditions will be reviewed. The focus will be on the energetic consequences of these adaptations as reflected by the growth yield value for the energy source (Y energy source). It will be illustrated that Y energy source values can be dramatically lowered as a result of incomplete oxidation of the energy source (overflow metabolism), bypassing potential sites of energy conservation (uncoupling) or catabolic cycles that have no other apparent effect than the hydrolysis of ATP (futile cycles). Thus, it is concluded that adaptation to low nutrient conditions aims at maintaining high metabolic fluxes at low nutrient concentrations at the cost of a loss in the energetic efficiency of the overall metabolism.

Adaptation, Physiological↗
PubMed1996

Pyrroloquinoline quinone, a chemotactic attractant for Escherichia coli.

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.

Bacterial Proteins↗
PubMed1994

Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775.

Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h-1 to 0.5 h-1, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.

Acetates↗
PubMed1994

The energetics of bacterial growth: a reassessment.

The growth yield of microbial cultures can be used to estimate the efficiency of energy generation during a fermentation or respiration. In the past, the assessment of this efficiency in organisms carrying out a respiration has been the subject of many heated debates. This has partly been caused by the complexity of microbial respiratory chains. Strains of Escherichia coli specifically modified in their respiratory chain have been used recently to re-evaluate the energetic efficiency of the bacterial respiration using chemostat cultures. The different strains indeed show different growth efficiencies. The physiological significance of energetically less-efficient branches of the respiratory chain is discussed.

Adenosine Triphosphate↗
PubMed1993

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecalis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Aerobiosis↗
PubMed1993

Energy conservation by pyrroloquinoline quinol-linked xylose oxidation in Pseudomonas putida NCTC 10936 during carbon-limited growth in chemostat culture.

When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents. Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate. No other products were formed from this pentose, nor was it incorporated into biomass. The presence of xylose in these cultures resulted in higher Yglucose and Ylactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose. As the Y0 values for growth on glucose or on lactate alone were equal to the Y0 values for growth with xylose as co-substrate, it is concluded that for glucose- or lactate-limited growth energy conservation by PQQH2 oxidation is as efficient as by NADH2 oxidation.

Bacterial Proteins↗
PubMed1993

Gluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5-5.5) the majority (70-90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.

Aerobiosis↗
PubMed1993

The role of lipoic acid in product formation by Enterococcus faecalis NCTC 775 and reconstitution in vivo and in vitro of the pyruvate dehydrogenase complex.

The role of the pyruvate dehydrogenase complex (PDC) in the formation of different fermentation products by Enterococcus faecalis was studied. This organism was grown on a semi-defined medium under various conditions in the presence or absence of lipoic acid, an essential cofactor of the enzyme complex. When grown on a medium without added lipoic acid, a very low activity, both in vivo and in vitro, of the PDC was observed. When pyruvate served as the energy source, lipoic acid was found to be essential for growth under anaerobic conditions at low culture pH values. The presence of lipoic acid in the culture medium had a marked effect on the production of acetoin: in the presence of lipoic acid, acetoin was produced only when the intracellular pyruvate concentration was relatively high, whereas in the absence of lipoic acid, acetoin was a common product. Under potassium-limited conditions, lactate was the main product and culture pH significantly affected the bacterial dry weight. After instantaneous addition of lipoic acid to a glucose+pyruvate-limited chemostat culture, an immediate activation of the PDC took place as deduced from the change in fermentation pattern. Reconstitution of the PDC by the addition of lipoic acid was also possible in cell-free extracts, although pre-incubation with ATP and lipoic acid for 90 min was necessary for maximal activation. The effects of an active PDC on product formation and the physiological role of the complex under anaerobic growth conditions are discussed.

Acetoin↗
PubMed1993

pH and Glucose Profiles in Aggregates of Bacillus laevolacticus.

Size distributions and glucose and pH profiles of aggregates of the d-(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.

Journal Article↗
PubMed1993

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Cell Division↗
PubMed1992

Physiological and energetic aspects of bacterial metabolite overproduction.

This review attempts to provide a rational explanation for microbial metabolite over-production, a phenomenon manifest with many wild-type and mutant organisms. After analysing the relationships between catabolic and anabolic processes within the growing cell (which point to the presence of sufficient kinetic control elements to ensure a stringent coupling between the two), consideration is given to mechanisms that might allow the rate of catabolism to be varied independently of the rate of anabolism. It is in this latter context that an excretion of specific metabolites appears to play a key role.

Bacteria↗
PubMed1992

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.

Acetyltransferases↗
PubMed1992

Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.

Acetates↗
PubMed1992

Anaerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture: involvement of the pentose phosphate pathway.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.

Adenosine Triphosphate↗
PubMed1991

Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli.

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or DL-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption. These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the Kdp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.

Acetates↗
PubMed1991

Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.

Adenosine Triphosphate↗
PubMed1990

The role of futile cycles in the energetics of bacterial growth.

In this contribution we describe the occurrence of futile cycles in growing bacteria. These cycles are thought to be active when organisms contain two uptake systems for a particular nutrient (one with a high, the other with a low affinity for its substrate). The high-affinity system is responsible for uptake of the nutrient, some of which is subsequently lost to the medium again via leakage through the low-affinity-system. A special futile cycle is caused under some growth conditions by the extremely rapid diffusion of ammonia through bacterial membranes. When the ammonium ion is taken up via active transport, the couple NH3/NH4+ will act as an uncoupler. This is aggravated by the chemical similarity of the potassium and the ammonium ion, which leads to ammonium ion transport via the Kdp potassium transport system when the potassium concentration in the medium is low. Other examples of futile cycles, such as those caused by the production of fatty acids by fermentation, are briefly discussed.

Adenosine Triphosphatases↗