M. J. Teixeira de Mattos

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal conditions.

Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

ABSTRACT Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in bakers yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.

Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces

ABSTRACT Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome. Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

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.

Respiration of Escherichia coli Can Be Fully Uncoupled via the Nonelectrogenic Terminal Cytochrome bd-II Oxidase

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

Quantitative Analysis of the High Temperature-induced Glycolytic Flux Increase in Saccharomyces cerevisiae Reveals Dominant Metabolic Regulation

A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperature-induced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate. Growth at increased temperature (38 °C versus 30 °C) resulted in a strongly increased glycolytic flux, accompanied by a switch from respiration to a partially fermentative metabolism. We observed an increased flux through all enzymes, ranging from 5- to 10-fold. We quantified the contributions of direct temperature effects on enzyme activities, the gene expression cascade and shifts in the metabolic network, to the increased flux through each enzyme. To do this we adapted flux regulation analysis. We show that the direct effect of temperature on enzyme kinetics can be included as a separate term. Together with hierarchical regulation and metabolic regulation, this term explains the total flux change between two steady states. Surprisingly, the effect of the cultivation temperature on enzyme catalytic capacity, both directly through the Arrhenius effect and indirectly through adapted gene expression, is only a moderate contribution to the increased glycolytic flux for most enzymes. The changes in flux are therefore largely caused by changes in the interaction of the enzymes with substrates, products, and effectors.

Alternative routes to biofuels: Light-driven biofuel formation from CO2 and water based on the ‘photanol’ approach

For a sustainable energy future, the development of efficient biofuel production systems is an important prerequisite. Here we describe an approach in which basic reactions from phototrophy are combined in single organisms with key metabolic routes from chemotrophic organisms, with C(3) sugars as Glyceraldehyde-3-phosphate as the central linking intermediate. Because various metabolic routes that lead to the formation of a range of short-chain alcohols can be used in this approach, we refer to it as the photanol approach. Various strategies can be explored to optimize this biofuel production strategy.

Metabolic and energetic aspects of the growth of Klebsiella aerogenes NCTC 418 on glucose in anaerobic chemostat culture

Klebsiella aerogenes NCTC 418 was cultured anaerobically in chemostat cultures (pH 6.8; 35° C) under carbon, phosphate-, ammonia-, sulphate- and potassium-limited conditions with glucose as the sole carbon- and energy source. The rates of uptake of glucose and excretion of fermentation products were quantitatively determined and carbon, hydrogen- and oxygen balances were constructed with recoveries better than 90%.It was found that under glucose-limited conditions the utilization of the carbon source was the most efficient. Under these conditions the highest YGLU was obtained whilst virtually all glucose was fermented to acetate and ethanol and formate plus carbon dioxide. Under all glucose-sufficient conditions a branched fermentation was observed with acetate, ethanol, formate plus carbon dioxide, D-lactate, succinate and 2,3-butanediol as end-products. The lower YGLU values for these cultures appeared to be a consequence of both a decreased YATP and a decreased efficiency of ATP synthesis from the dissimilation of glucose. In contrast to aerobic cultures, significant differences in fermentation patterns were not observed between the various glucose-sufficient cultures.The fermentation patterns of glucose-sufficient cultures appeared to be influenced by the specific growth rate. For all these cultures a lowering of the dilution rate resulted in a smaller fraction of glucose being fermented to acetate and a greater fraction being fermented to 2,3-butanediol. Relatively much glucose was fermented to D-lactate when the organisms were grown at a low dilution rate under sulphate-limited conditions.Anaerobic, glucose-limited cultures were found to be able to increase their specific uptake rate of glucose instantaneously when pulsed with glucose. The extra glucose taken up was fermented mainly to D-lactate whilst no increase in the specific production rate of acetate and ethanol occurred. The excreted D-lactate probably was not formed from pyruvate, by the NADH-linked lactate dehydrogenase, but via the methylglyoxal bypass. It is suggested that by invoking this bypass reaction K. aerogenes can metabolically uncouple ATP synthesis from glucose dissimilation, and herein may lie the physiological significance of this reaction sequence.

Energetic consequences of multiple K+ uptake systems in Escherichia coli

The energetics of growth of Escherichia coli FRAG 1 under potassium-limited growth conditions and with glucose as sole carbon and energy source were studied in the chemostat and compared with those of a mutant, FRAG 5, defective in the high-affinity potassium uptake system. The steady-state concentration of biomass decreased with increasing growth rate and was the same in both parent and mutant. For each growth rate, the rate of production of ATP was higher in the parent than the mutant strain. Under potassium-limited conditions, FRAG 1 has at least two potassium uptake systems, an inducible high-affinity uptake system and a constitutive low-affinity uptake system (Rhoads, D.B., Waters, F.B. and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Apparently, the presence of the high-affinity uptake system in the parent leads to an energy drain. We suggest that this energy drain is due to futile cycling of potassium ions. On the basis of a mosaic non-equilibrium thermodynamic description of bacterial growth, it is concluded that the growth behaviour under potassium limitation corresponds to that expected for a catabolite limitation.

Metabolic uncoupling of substrate level phosphorylation in anaerobic glucose-limited chemostat cultures of Klebsiella aerogenes NCTC418

Klebsiella aerogenes NCTC418 was cultured anaerobically under glucose-limited conditions in chemostat cultures at various growth rates, ranging from 0.13 h-1 to 0.82 h-1. It was found that the specific uptake rate of glucose varied linearly with the growth rate and that under these conditions glucose was fermented solely to acetate and ethanol plus CO2+H2 and formate.When steady-state cultures were pulsed with cell saturating concentrations of glucose, the specific glucose aptake rate increased immediately and substantially. However, at steady-state growth rates lower than 0.5 h-1, this increase was not accompanied by a change in the growth rate, in contrast to cultures growing at higher rates. It was found that relief of the glucose limitation resulted in a shift in fermentation pattern: at the lower growth rates 50% or more of the extra glucose taken up was fermented to D-lactate.Incubation experiments with sonified cells revcaled that K. aerogenes possessed all the enzymes needed to convert dihydroxyacetone phosphate to methylglyoxal and subsequently to D-lactate, and that the rate at which this overall conversion occurred in vitro was in close agreement with the production rate of D-lactate in vivo. Moreover, it was found that the activities of the enzymes of the methylglyoxal bypass were dependent on the imposed growth rate. At higher growth rates, where cells possessed the potential to increase their growth rate immediately, the activity of methylglyoxal synthase was relatively low.it could be shown that, under low growth rate conditions, the uncoupling effect of the methylglyoxal bypass was highly effective and that, as a consequence thereof, a significant increase in the uptake rate of the energy source was accompanied by only a marginal increase in the rate at which ATP was synthesized.