J.L. Snoep

Transduction of Intracellular and Intercellular Dynamics in Yeast Glycolytic Oscillations

Under certain well-defined conditions, a population of yeast cells exhibits glycolytic oscillations that synchronize through intercellular acetaldehyde. This implies that the dynamic phenomenon of the oscillation propagates within and between cells. We here develop a method to establish by which route dynamics propagate through a biological reaction network. Application of the method to yeast demonstrates how the oscillations and the synchronization signal can be transduced. That transduction is not so much through the backbone of glycolysis, as via the Gibbs energy and redox coenzyme couples (ATP/ADP, and NADH/NAD), and via both intra- and intercellular acetaldehyde.

Construction and characterization of an effector strain of streptococcus mutans for replacement therapy of dental caries.

ABSTRACT An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make theStreptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of theldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF fromZymomonas mobilis in place of the deleted ldhORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic- and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10−3) or by screening cultures grown in vitro, where no revertants were observed among 105 colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.

DNA supercoiling depends on the phosphorylation potential in Escherichia coli

ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well‐defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free‐energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.

Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1 ‐ 2.9 mM, consistent with the function of Gif as a low‐affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf‐dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Gif for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.

Branched-Chain α-Keto Acid Catabolism via the Gene Products of the bkd Operon in Enterococcus faecalis: a New, Secreted Metabolite Serving as a Temporary Redox Sink

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.

Glucose and the ATP paradox in yeast.

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.

The Extent to Which ATP Demand Controls the Glycolytic Flux Depends Strongly on the Organism and Conditions for Growth

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.

DNA Supercoiling by Gyrase is Linked to Nucleoid Compaction

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 μm cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.

Regulation of energy source metabolism in streptococci

availability of nutrients. Thus, if we wish to understand the From the classical microbiological literature one would conbehaviour of organisms in natural environments it is crucial clude that energy source metabolism of streptococci and that we pay more attention to the phenotypic variability of related organisms is relatively simple: acid (lactic acid) is microbes, because such studies will provide us with more produced from glucose. This was considered for a long time insight into the physiological relevance of the different strucas the dominant product. Occasionally reference was made to tural and metabolic properties of microbes. In this review we the production of minor quantities of other products (acetate, will show that many environmental factors exert a profound ethanol and carbon dioxide) under special growth conditions. influence on energy source metabolism in streptococci. The ability to produce gas from glucose was also used as a