Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).     

Effects of growth conditions on mitochondrial morphology inSaccharomyces cerevisiae

Effects of growth conditions on mitochondrial morphology were studied in livingSaccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 µmole.min^–1.g weight^–1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology inS. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.     

Physiological state, growth mode, and oxidative stress play a role in Cd(II)-mediated inhibition of Nitrosomonas europaea 19718

The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of Nitrosomonas europaea strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and amoA expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [K(i)]) in exponential batch cultures was observed. Long-term inhibition levels (based on K(i) estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in amoA expression) and starvation (the retention of 16S rRNA levels) in N. europaea cultures were observed. amoA expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the amoA response was consistent with that of the sOUR, the amoA response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of N. europaea was attributed partially to oxidative stress.     

Quantitative description of microbial growth in a batch culture depending on the physiologic state of inocula]

The dynamics of biomass production and the respiration rate of five microorganisms grown as batch cultures were studied in detail. Cell suspensions with a known physiological state, i.e. chemostat cultures grown at a particular D value, as well as quasi steady-state populations cultivated with slow feeding and long energy-source starvation were used as inocula. The microorganisms were arbitrary subdivided into two groups. The biomass of Pseudomonas fluorescens, Bacillus subtilis and Debaryomyces formicarius accumulated smoothly with a monotonic rise of the specific growth rate to the upper level of microns. A distinct inverse correlation was established between the duration of the lag phase and the specific growth rate of the inoculum. Arthrobacter globiformis and Lipomyces tetrasporus were characterised by biphase growth referred to as false diauxia: glucose was accumulated without its oxidation during the first phase, and actual growth (just as in the first group of microorganism) occurred during the second phase. Most of the results are satisfactorily described by a simplified modification of the synthetic chemostat model.     

Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.     

Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady-state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady-state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. (c) 1992 John Wiley & Sons, Inc.     

Effect of glucose analog supplementation on metabolic flux distribution in anaerobic chemostat cultures of Escherichia coli

Previous work in our laboratories investigated the use of methyl alpha-glucoside (alpha-MG), a glucose analog that shares a phosphotransferase system with glucose, to modulate glucose uptake and therefore reduce acetate accumulation. The results of that study showed a significant improvement in batch culture performance and a reduction in acetate excretion without any significant effect on the growth rate in complex medium. The current study investigates the effect of supplementing the culture medium with the glucose analog alpha-MG on the metabolic fluxes of Escherichia coli under anaerobic chemostat conditions at two different dilution rates. Anaerobic chemostat studies utilizing complex media supplemented with glucose or glucose and alpha-MG at dilution rates of 0.1 and 0.4 h(-1), were performed, and the metabolic fluxes were analyzed. It was found that the addition of the glucose analog alpha-MG has an effect on the specific production rate of various extracellular metabolites. This effect is slightly greater at the higher dilution rate of 0.4 h(-1). However, the glucose analog does not cause any major shift in the central metabolic patterns. It was further observed that alpha-MG supplementation does not result in the reduction in specific acetate synthesis rate in anaerobic chemostat cultures. These results emphasize the importance of testing different strategies for metabolic manipulation under the actual operating conditions.     

Proteomic studies of diauxic lag in the differentiating prokaryote Streptomyces coelicolor reveal a regulatory network of stress-induced proteins and central metabolic enzymes

Bacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH4+, formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic- and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http://proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold- and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons. Although stress stimulons presumably provide protection during environmental- or starvation-induced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor.     

In vivo kinetics of primary metabolism in Saccharomyces cerevisiae studied through prolonged chemostat cultivation

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics of Saccharomyces cerevisiae. S. cerevisiae was grown in carbon-limited aerobic chemostats for 70-95 generations, during which multiple steady states were observed, characterized by constant intracellular fluxes but significant changes in intracellular metabolite concentrations and enzyme capacities. We provide evidence for two relevant kinetic mechanisms for sustaining constant fluxes: in vivo near-equilibrium of reversible reactions and tight regulation of irreversible reactions by coordinated changes of metabolic effectors. Using linear-logarithmic kinetics, we illustrate that these multiple steady-state measurements provide linear constraints between elasticity parameters instead of their absolute values. Upon perturbation by a glucose pulse, glucose uptake and ethanol excretion in prolonged cultures were remarkably lower, compared to a reference culture perturbed at 10 generations. Metabolome measurements during the transient indicate that the differences might be due to a reduced ATP regeneration capacity in prolonged cultures.