Enzyme induction and repression in Arthrobacter crystallopoietes

Summary Catabolic effects which exert control over the inducible synthesis of three enzymes in Arthrobacter crystallopoietes involve at least three different mechanisms: interference with inducer transport, severe catabolite repression, and transient repression. The rate of histidase induction by histidine is reduced by incubation of the cells with succinate or glucose. The maximum effect of succinate, 67% reduction in histidase production, occurs only after 100 min of incubation with succinate. At least 3h of incubation are required for the maximum effect of glucose (31% reduction in enzyme induction). Both succinate and glucose inhibit histidine transport. Cyclic adenosine 3,5-monophosphate (cyclic AMP), at 10^-7 M, slightly stimulates the induction of histidase in cultures both with or without succinate. No conditions were found in which cyclic AMP abolishes the effect of succinate. Induction of l-serine dehydratase by glycine is severely and permanently repressed by glucose and to a lesser extent by citrate. Glucose does not affect glycine uptake. Succinate, fumarate, and aspartate, which are all better substrates than glucose or citrate for growth of A. crystallopoietes, have no effect on l-serine dehydratase induction. Induction and repression of l-serine dehydratase are not affected by cyclic AMP. Synthesis of isocitrate lyase after addition of acetate is unaffected by glucose but is severely repressed by succinate or fumarate. Aspartate and glutamate cause a transient repression of enzyme synthesis after which synthesis proceeds at the control rate. The ability to transport acetate is inducible. Development of this capacity in the presence of acetate is not affected by succinate or glutamate. Cyclic AMP has no effect on enzyme production or repression. A. crystallopoietes takes up radioactive cyclic AMP and has at least one of the enzymes of cyclic AMP metabolism, adenyl cyclase.     

Intermediate metabolism inTrypanosoma cruzi

Epimastigotes ofTrypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of NH₃, and glucose with production of reduced catabolites, chiefly succinate andl-alanine, even under aerobic conditions. This aerobic fermentation of glucose is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate orl-alanine, andl-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic -hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.     

Heterotrophic growth of Thiobacillus A2 on sugars and organic acids

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and -linked disaccharides, but not on -linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occurred on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.     

Growth conditions and temperature-dependent substrate specificity of two extremely thermophilic bacteria

Summary Conditions for cultivating two extremely thermophilic bacteria, isolated from the hot springs of Yellowstone National Park, are described. One of these strains, Thermus aquaticus, can be grown on either succinate or pyruvate as the best substrates at 78° C. Acetate, glucose, and sucrose can also be utilized at this temperature. The temperature optimum was found to be 70° C, but the bacterium can be adapted to grow on succinate or pyruvate at 80° C. The other strain, YT-G has its growth optimum at 80° C and the maximum temperature was found to be 84° C. At this temperature pyruvate is the only substrate which gives good results, while glucose cannot be used as a carbon source. At 70° C, however, the yields obtained with glucose as a substrate are better than those with pyruvate at 80° C.Experiments with C¹⁴-labelled glucose have shown that the inability to utilize glucose at 80° C is not due to an inactivation of the initial steps of the glycolytic pathway. Phosphorylated sugars and a compound corresponding to -glycerophosphate were found to be formed, the latter being accumulated as a side product of normal glycolysis. The enzymes leading to this product, and those which are involved in the conversion of pyruvate were found to be functioning at 80° C, while intermediate enzymes of the glycolytic pathway are assumed to be less heat resistant, thus blocking the utilization of glucose at this temperature. The ability of strain YT-G to grow on glucose is, however, promptly resumed if the temperature is lowered.Lysozyme treatment was found to lead to a complete conversion of T. aquaticus cells to spheroplast while cells of strain YT-G are only slightly altered by this procedure.     

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes malic enzyme, NAD(P)H: ferredoxin oxidoreductase, pyruvate: ferredoxin oxidoreductase, hydrogenase, acetate: succinate CoA transferase and succinate thiokinase leading to the formation of H₂, CO₂, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O₂-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role-especially in zoospores — as H₂-evolving, ATP-generating and O₂-scavenging organelles.Abbrevations DTT Dithiotreitol - PEP Phosphoenol pyruvate     

Effect of the level of the carbon source on the activity of some lytic enzymes released during autolysis of Aspergillus niger

Changes in the activity of -N-acetylglucosaminidase, chitinase, invertase, esterases, glucanases and phosphatases liberated into the culture fluid were followed during the autolytic phase of growth of Aspergillus niger on media with various initial levels of the carbon source. The general pattern was of an accumulation of these lytic enzymes in the culture fluid during autolysis, but some enzymes reached maximum activity and then declined. The initial level of the carbon source affected the enzyme pattern during autolysis. Maximum activity for the various enzymes was always observed either for the lowest initial level of carbon or the highest (3.5 mM glucose, 111 mM glucose). The highest specific activities were those for exopolygalacturonidase (500 mU/mg at 3.45 mM glucose), and for -amylase (about 500 mU/mg at 3.45 mM glucose). Cellulase, chitinase and esterase showed the weakest activity. Acid phosphatase was most active (about 200 mU/mg) at 3.45 mM initial glucose, whereas alkaline phosphatase was most active (45 mU/mg) at 111 mM glucose, both during the autolytic phase of growth.     

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, Q _O2, remained between 14–18 mmol O₂ h^-1 g dwt^-1 and the maximum growth rate varied from 0.22 h-¹ on acetate to 0.77 h^-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µ_crit, the growth yield Y_XO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of m_ATP < 4 mmol ATP h^-1 g dwt^-1 determined from A-stat cultivation data was low compared with the calculated unproductive hydrolysis of ATP (64–103 mmole ATP g dwt^-1).     

The Effect of Succinate on Respiration,Transamination, and Pyruvate Formation in Cells of the Yeast <Emphasis Type="Italic">Dipodascus magnusii</Emphasis>

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

Systematic engineering of pentose phosphate pathway improves <Emphasis Type="Italic">Escherichia coli</Emphasis> succinate production

Background

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production.

Results

Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge.

Conclusions

In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

     

Expression of Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli without Affecting Glucose Uptake

Plasmids carrying the pyc gene from Rhizobium etli were used to express pyruvate carboxylase in Escherichia coli. Results of batch fermentations of a wild-type E. coli (MG1655), this wild-type with the pUC18 cloning/expression vector (MG1655/pUC18) and this wild-type carrying the pyc gene (MG1655/pUC18-pyc) were compared in glucose-limited medium. The results indicate that the final succinate concentration upon complete glucose utilization was increased from 1.18 g/L to 1.77 g/L by the expression of pyc, while the final succinate concentration in MG1655/pUC18 was slightly lower than in the parent strain. This increased succinate concentration came at the expense of lactate synthesis, whose final concentration decreased from 2.33 g/L to 1.88 g/L. The expression of pyc did not affect the maximum glucose uptake (2.17 g/Lh for MG1655 versus 2.47 g/Lh for MG1655/pUC18-pyc), but did decrease the maximum rate of cell mass production (0.213 g/Lh for MG1655, 0.169 g/Lh for MG1655/pUC18 and 0.199 g/Lh for MG1655/pUC18-pyc).     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

Glycogen in Methanothrix

An intracellular glycogen was purified and characterized from the acetoclastic bacteria Methanothrix str. FE, its average chain length was about 13 glucose residues. Acetyl-CoA was shown to be synthesized by the action of acetate thiokinase; in addition pyruvate synthase, phosphoenolpyruvate synthetase and enzymes of gluconeogenesis were detected in cell extracts. For glycogen synthase activity, both adenosine diphosphate glucose and uridine diphosphate glucose were used as glycosyl donors, apparent K _m were, respectively, 8 M for ADPGlc and 625 M for UDPGLe, at the opposite the V _m were the same for both precursors. This was in accordance with competition experiments and strongly suggested that only one glucosyl transferase was involved and that ADPGlc was the physiological glycosyl donor in Methanothrix str. FE. In addition branching enzyme activity (1-4-glucan-6-glucosyl transferase) was detected in cell extracts.Abbreviations ADPGlc adenosine diphosphate glucose - UDPGlc uridine diphosphate glucose     

Energy metabolism of some representatives of the Haemophilus group

The purpose of this investigation was to characterize the carbohydrate catabolism and the constellation of the respiratory chain components of Haemophilus influenzae RAMC 18 Bensted, H. parainfluenzae 1 Fleming, H. parainfluenzae 429 Pittman and H. aegyptius 180a Pittman. These strains represent several physiological types with respect to respiratory quinones and glucose catabolism.On addition of glucose or lactate to the complex growth medium a remarkable increase in cell mass was observed. Depending on the growth rate, carbohydrate degradation varied with the strains examined so that at the end of the exponential growth phase only small amounts of the supplements could be demonstrated.All strains were found to possess functional enzymes of Embden-Meyerhof-Parnas-, Entner-Doudoroff-pathways, hexosemonophosphate shunt, tricarboxylic acid cycle and gluconeogenesis with an extremely high activity of malate dehydrogenase.The concentration of cytochromes varied according to culture conditions. The cytochromes a₁, d, o and b+c were found to occur under aerobic conditions. In cells grown anaerobically in the presence of fumarate cytochromes a₁ and d could not be demonstrated. Under aerobic conditions preparations of H. parainfluenzae 1 Fleming exhibited an -maximum at 558 nm, whereas under anaerobic culture conditions with fumarate as terminal electron acceptor an -maximum at 552 nm occurred, suggesting different roles of b and c type cytochromes in aerobic and anaerobic electron transport to fumarate, respectively.     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources

Background

Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable).

Results

A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis.Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions.

Conclusions

The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1484-6) contains supplementary material, which is available to authorized users.     

d-arabinose metabolism byBacteroides fragilis strain 2044

During growth in the presence of 1-14C-d-arabinose,Bacteroides fragilis strain 2044 formed labeled succinic, acetic, and propionic acis. Degradation of the acids by the Schmidt reaction revealed that at least 89% of the succinate radioactivity was found in the methylene carbons and that 75% and 84% of the label in propionate and acetate were found in the noncarboxyl carbons of these molecules. No label was found in acetate, propionate, or succinate during growth of strain 2044 in the presence of 5-3H-d-arabinose. Strain 2044 converted radioactivity from 1- or 2-labeled glycolic acid and glycine to succinate by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons. Label from 1- or 2-labeled glycine and 2 but not 1-labeled glycolic acid was found in acetate. Uniformly labeled 14C-glyoxalate gave rise to labeled acetate, but not succinate.Bacteroides fragilis strain 2044 metabolizesd-arabinose by a mechanism involving a 32 cleavage of the molecule.     

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.     

Secretory enzyme production and conidiation of Aspergillus oryzae in submerged liquid culture

Summary The production of - and -galactosidases, proteinase and -amylase and also conidiation of Aspergillus oryzae were examined in liquid soybean meal culture. In a culture of soybean meal only, conidiation of the fungus was not induced and the production of the enzymes was not significant, although fungal growth was abundant. When phosphate was added to the medium at concentrations above 0.2 M, enzyme production was significantly increased and the cells formed conidiophores after enzyme production had attained maximum level. Increase in production of galactosidases was the most marked.K- or Na-salts other than phosphate were not effective stimulants for enzyme production, while conidiation was not induced under these growth conditions. Conidiation and production of enzymes were repressed by the addition of glucose or casamino acids to the soybean meal medium containing KH₂PO₄.Conidiation and enzyme production were also studied in modified Czapek media in which sucrose was replaced by other carbon sources. Lactose, lactulose, melibiose and polysaccharides composed of galactosyl linkage such as arabinogalactan induced both conidiation and production of the enzymes.     

Oxidative phosphorylation in Micrococcus denitrificans

P/O ratios were measured in membrane particles obtained from cells of Micrococcus denitrificans, while growing on different carbon sources. The membrane particles obtained from cells growing actively on glucose, succinate, ethanol and propanol as the carbon and energy sources catalyzed oxidative phosphorylation and yielded respective P/O ratios of 1.4, 1.2, 0.8, and 0.5 with NADH, and 0.8, 0.6, 0.6, and 0.5 with succinate as the electron donors. Not such a difference in P/O ratio is observed in intact resting cells grown with different carbon sources. It is concluded that the influence of the carbon source is probably directed towards the efficiency of oxidative phosphorylation in membrane particles and not in the growing cells.For the aerobic carbon source-limited chemostat cultures the following maximum growth yields were determined: 40.2 and 34.2 for succinate and oxgen, 41.7 and 36.5 for malate and oxygen, 81.4 and 39.4 for mannitol and oxygen, and 77.8 and 43.4 for gluconate and oxygen respectively. With a mathematical model (de K waadsteniet et al., in press) the P/O ratio was valued at 1.4–1.7. Y _ATP at =0.2 was valued at 8.7–10.9; Y _ATP ^max at 9.6–13.2 and m _e at 0.6–4.5 for the most precise experiment (gluconate-limited). The calculation of these growth parameters has been discussed.     

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)^-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO₂-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO₂-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO₂ release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)