The Pasteur effect and catabolite repression in an oxidative yeast,Kluyveromyces lactis

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.     

Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D.

Sensitivity to actinomycin D(AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.     

Dual effects of glucose on dicarboxylic acid transport in Kluyveromyces lactis.

The properties of succinate uptake in succinate-grown Kluyveromyces cells were examined. The rate of succinate transport at 15C exhibits an approximate V-max of 1.2 mumol times h-1 times mg-1 dry weight of cells and an apparent K-m of 18 muM. The uptake process appears to be tightly coupled to metabolism. L-Malate, fumarate, and alpha-ketoglutarate were the only other dicarboxylates tested, which were found to inhibit succinate transport. The aggreement between the order of inhibition of succinate transport by these dicarboxylates and their rates of uptake, as well as the competitive nature of the inhibition are all consistent with the existence of a common carrier system showing specificity for dicarboxylates of the TCA cycle. Cells transferred from succinate to glucose medium rapidly lose their ability to transport succinate. Glucose-grown cells also exhibit an inability to oxidize dicarboxylates or to use them for growth without a very long lag. The dicarboxylate uptake system, therefore, appears to be subject to a strong catabolite repression. The depression of the succinate transport system requires the presence of succinate, as well as low concentrations of glucose.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92–99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93–94% ee) compared with cells harvested in exponential phase (43–60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65–81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92-99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93-94% ee) compared with cells harvested in exponential phase (43-60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65-81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.     

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.     

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.     

An Escherichia coli mutant conditionally altered in respiratory chain components

Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components. Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-). Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*). The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate. Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions. The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation. R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate. R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25. The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E. coli genome. Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.     

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized -galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show supressed respiration of TCA cycle compounds; -galactosidase was not synthesized because the mutant lacked a functional PTS. Cyclic adenosine-3, 5-monophosphate did not reverse the repression by glucose of SEA or -galactosidase synthesis in glucose-grown S. aureus 196E. An active PTS appears to be necessary to demonstrate glucose (catabolite) repression in S. aureus.Abbreviations SEA staphylococcal enterotoxin A - SEB staphylococcal enterotoxin B - SEC staphylococcal enterotoxin C - PTS phosphoenolpyruvate phosphotransferase system - CAS casamino acids salts medium - TCA tricarboxylic acid cycle     

Some Factors Governing Respiration,Glucose Metabolism and Iodoacetate Sensitivity in Tetrahymena pyriformis*

SYNOPSIS. The physiologic response of Tetrahymena pyriformis W to glucose, as measured by the respiratory rate in a buffered suspension, was found to be altered by exposure of the culture to this carbohydrate in the peptone-based growth medium. Glucose-grown cells had an elevated respiratory rate in the presence of glucose in the Tris-buffered suspension, while cells not grown with the glucose supplement required sodium and potassium for the response. Orthophosphate elevated the rate of oxygen consumption independently of the glucose effect. The endogenous respiratory of glucose and non-glucose cultured cells were significantly diffe The 2 types of cells responded differently to the cations, sod and potassium, and were not identical in sensitivity to iodoacet Iodoacetate poisoning was found to be due to inhibition of “uncompetitive” type, potentiated by glucose, orthophosphate, the absence of potassium.     

beta-Glucose-1-Phosphate, a Possible Mediator for Polysaccharide Formation in Maltose-Assimilating Lactococcus lactis

Homolactic fermentation of glucose and heterolactic fermentation of maltose with Lactococcus lactis 65.1 were confirmed. When moles of glucose were compared, the uptake rates of the two carbon sources were similar. The intracellular concentration of fructose-1,6-diphosphate (FDP) in maltose-assimilating cells was half of that in glucose-assimilating cells. Similarly, formation of FDP and lactate from maltose by extracts of maltose-grown cells was half of that formed from glucose by extracts of glucose-grown cells, indicating a difference in the utilization of the two carbon sources for energy metabolism. Concentrations of adenine nucleotides were similar in both types of cells. Glucose-1-phosphate was found in extracts of maltose-grown cells given maltose and, in addition, an inducible and low beta-specific phosphoglucomutase activity was observed. beta-Glucose-1-phosphate was not metabolized by cell extracts to either FDP or lactate, suggesting an alternative metabolic route. The amount of [C]maltose incorporated into the cell material of maltose-grown cells was four times greater than that of [C]glucose incorporated into the cell material of glucose-grown cells. The intracellular concentration of UTP was lower in maltose-assimilating cells than in glucose-assimilating cells. Cells grown on maltose were more spherical and less fragile than cells grown on glucose.     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

The Effect of Nonanal on Dipodascus aggregatus IV. Studies with Different Carbon Sources

Nonanal (80 μ) in ethanolic solution stimulated the growth of Dipodascus aggregatus with fructose (55.5 mM) as carbon source (inoculum grown with fructose or glucose). If the inoculum had been grown with galactose, neither growth with galactose nor growth with glucose was affected by nonanal. If the inoculum had been grown with glucose, growth with galactose was weakly. stimulated. —Growth with galactose (galactose-grown inoculum) was strongly stimulated by nonanal if xylose at a low concentration (0.53 mM) was added. — The oxygen uptake of glucose grown cells with glucose as substrate was stimulated by 200 μM nonanal in the absence of ethanol. The respiratory activity of galactose-grown cells was also stimulated with galactose as well as with glucose as substrate. In the absence of exogenous substrate the oxygen uptake of glucose-grown cells was weakly stimulated by nonanal whereas that of galactose-grown cells was strongly stimulated.     

Endogenous metabolism of Azotobacter agilis

Sobek, J. M. (University of Southwestern Louisiana, Lafayette), J. F. Charba, and W. N. Foust. Endogenous metabolism of Azotobacter agilis. J. Bacteriol. 92:687-695. 1966-Ribonucleic acid, deoxyribonucleic acid, cellular carbohydrate, and the cold trichloroacetic acid and acidic alcohol fractions of the cell do not appear to function as endogenous reserves for Azotobacter agilis. The immediate endogenous reserve of cells grown on glucose, acetate, or succinate was poly-beta-hydroxybutyric acid (PHB). Viability of the cells during starvation was dependent upon the initial levels of PHB and the growth substrate. Cells with high initial PHB levels survived longer than cells with lower levels. Cells from succinate-grown cultures had lower PHB levels than cells from glucose-grown cultures, but were capable of maintaining their viability longer. Cellular protein may also serve as a secondary endogenous reserve substrate for this organism.     

Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase

Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.