Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Growth in mixed substrates

1. The type of metabolism adopted by Pseudomonas oxalaticus during growth on a variety of carbon sources was studied. 2. The only substrate upon which autotrophic growth was observed is formate. 3. In mixtures of formate and those substrates upon which the organism can grow faster than on formate, e.g. succinate, lactate or citrate, heterotrophic metabolism results. 4. In mixtures of formate and those substrates upon which the organism can grow at a similar rate to that on formate, e.g. glycollate or glyoxylate, the predominant mode of metabolism adopted is heterotrophic utilization of the C₂ substrate coupled with oxidation of formate as ancillary energy source. 5. P. oxalaticus grows on oxalate 30% slower than on formate. In mixtures of formate and oxalate, the predominant mode of metabolism adopted is autotrophic utilization of formate coupled with oxidation of oxalate as ancillary energy source. 6. In mixtures of formate and those substrates upon which the organism grows at a much lower rate than on formate, e.g. glycerol and malonate, the predominant mode of metabolism adopted is autotrophic utilization of formate. 7. It is concluded that synthesis of the enzymes involved in autotrophic metabolism is controlled by a combination of induction and metabolite repression.     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Metabolic regulation in Pseudomonas oxalaticus OX1

Diauxic growth of Pseudomonas oxalaticus was observed on a mixture of formate and oxalate in batch cultures. In the first phase of growth only formate was used. The capacity to oxidize oxalate appeared during the lag phase of 2–4 h after the exhaustion of formate and was followed by a second phase of growth on oxalate. The rate of autotrophic ¹⁴CO₂ fixation measured in washed cell suspensions decreased markedly in this second growth phase on the addition of oxalate. In mixtures of formate with acetate, glyoxylate or glycollate, simultaneous utilization of both substrates was observed. During growth on acetate plus formate formate-oxidizing capacity remained low. With low acetate concentrations, sufficient formate remained after the exhaustion of acetate to support a second growth phase on formate. This phase followed a 1.5–2 h lag, during which formate-oxidizing capacity increased and the Calvin cycle enzymes were synthesized. In mixtures of formate with glyoxylate or glycollate, the formate-oxidizing capacity was high, formate was oxidized rapidly, and no second growth phase was seen. In these latter mixtures high activities of a membrane-bound, phenazine methosulphate/2,6-dichlorophenolindophenollinked formate dehydrogenase and low activities of the soluble NAD-linked formate dehydrogenase were detected. The synthesis of ribulose-1,5-diphosphate carboxylase was totally repressed during growth on formate plus glycollate and partially repressed on formate plus glyoxylate. The regulation of Calvin cyclus enzymes in Pseudomonas oxalaticus is discussed.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of oxalate and formate in continuous culture

Pseudomonas oxalaticus was grown in carbon- and energy-limited continuous cultures either with oxalte or formate or with mixtures of these substrates. During growth on the mixtures, simultaneous utilization of the two substrates occurred at all dilution rates tested. Under these conditions oxalate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and the ratio of oxalate and formate in the medium reservoir. At a fixed oxalate/formate ratio repression was greatest at intermediate dilution rates, whereas derepression occurred at both low and high dilution rates. Progressive depression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation at low dilution rates was attributed to the decreasing concentration of intracellular repressor molecule(s), parallel to the decreasing concentration of the growth-limiting substrates in the culture. To account for the derepression at higher dilution rates, it is proposed that the rate of oxalyl-CoA production from oxalate limits the supply of metabolic intermediates and that additional energy and reducing power generated from formate drains the pools of metabolic intermediates sufficiently to lower the intracellular concentration of the repressor(s). During growth of Pseudomonas oxalaticus on the heterotrophic substrate oxalate alone, at dilution rates below 10% of the maximum specific growth rate, derepression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation was observed to a level which was 50% of that observed during growth on formate alone at the same dilution rate. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO₂ fixation via the Calvin cycle is regulated by a repression/derepression mechanism and that the contribution of autotrophic CO₂ fixation to the biosynthesis of cell material in this organism is mainly controlled via the synthesis of these enzymes.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

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.     

Fermentation of L-tartrate by a newly isolated gram-negative glycolytic bacterium

Enrichments on L-tartrate from a freshwater lake sediment yielded a pure culture of anaerobic bacterium designated strain 16Lt1. The rod-shaped organism was motile, did not form spores, and had a gram-negative wall structure. No cytochromes were detected. The mol % G+C of the DNA was 58. The new strain was microaerotolerant, and grew optimally at 30°C and neutral pH in freshwater medium. A wide range of carbohydrates was fermented, with formate, acetate, ethanol, lactate and succinate being the end-products detected. L-tartrate and citrate were fermented to formate, acetate and CO₂. L-tartrate was fermented by the dehydratase pathway, and glucose by the Embden-Meyerhof-Parnas pathway. Fumarate was reduced, but nitrate, sulfate, sulfur and thiosulfate were not used as terminal electron acceptors. Glucose metabolism was constitutive, whereas L-tartrate-degrading activity was inducible. When glucose and L-tartrate were both present as substrates, growth was diauxic with glucose being metabolized first. The growth rate and growth yield were higher on glucose than on L-tartrate. Strain 16Lt1 has been deposited with the Deutsche Sammlung von Mikroorganismen as Bacteroides sp. DSM6268.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of acetate and formate in continuous culture

Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the inflowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10h^-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (S_R=30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Energy production and growth of Pseudomonas oxalaticus OX1 on oxalate and formate

The efficiency of oxidative phosphorylation in Pseudomonas oxalaticus during growth on oxalate and formate was estimated by two methods. In the first method the amount of ATP required to synthesize cell material of standard composition was calculated during growth of the organism on either of the two substrates. The [Y _ATP ^max ] theor. values thus obtained were 12.5 and 6.5 for oxalate and formate respectively, if the assumption were made that no energy is required for transport of oxalate or carbon dioxide. When active transport of oxalate requiring an energy input equivalent to 1 mole of ATP per mole of oxalate was taken into account, [Y _ATP ^max ]theor. for oxalate was 9.4. True Y _ATP ^max values were derived from these data on the assumption that the energy produced in the catabolism of Pseudomonas oxalaticus is used with approximately the same efficiency as in a range of other chemoorganotrophs. P/O ratios were calculated using the equation P/O=Y _O/Y _ATP. The data for Y _O and m _e required for these calculations were obtained from cultures of Pseudomonas oxalaticus growing on oxalate or formate in carbon-limited continuous cultures. The P/O ratios calculated by this method were, for oxalate, 1.3 (or 1.0 if active transport were ignored), and for formate, 1.7.In the second method the stoicheiometries of the respiration-linked proton translocations with oxalate and formate were measured in washed suspensions of cells grown on the two substrates. The H⁺/O ratios obtained were 4.3 with oxalate and 3.9 with formate. These data indicate the presence of two functional phosphorylation sites in the electron transport chain of Pseudomonas oxalaticus during growth on both substrates. A comparison of the P/O ratio on oxalate obtained with the two methods indicated that the energy requirement for active transport of oxalate has a major effect on the energy budget of the cell; about 50% of the potentially available energy in oxalate is required for its active transport across the cell membrane. Translocation of formate requires approximately 25% of the energy potentially available in the substrate. These results offer an explanation for the fact that molar growth yields of Pseudomonas oxalaticus on oxalate and formate are not very different.Abbreviations PMS phenazinemethosulphate - DCPIP 2,6-dichlorophenolindophenol - TMPD N,N,N,N-tetramethyl-1,4-phenylene-diamine dihydrochloride - SD standard deviation - PEP Phosphoenol-pyruvate     

Control of diauxic growth of Azotobacter vinelandii on acetate and glucose.

Batch cultures of Azotobacter vinelandii were inoculated with cells pregrown on either acetate or glucose. When they were subsequently grown on a mixture of acetate and glucose, typical diauxic growth was observed, with preferential uptake of acetate in the first and glucose in the second phase of growth. Extracts from acetate-pregrown cells exhibited high acetate kinase activity in the first phase of growth. This activity decreased and activities of the two glucose enzymes glucose 6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase increased in the second phase. Extracts from glucose-pregrown cells exhibited high initial activities of the two glucose enzymes, which decreased while acetate kinase activity increased in the first phase of growth. Again, in the second phase, activities of the two glucose enzymes increased and acetate kinase activity decreased. In any case, isocitrate dehydrogenase activity varied only slightly and unspecifically. The differences in enzyme activity and the constancy of isocitrate dehydrogenase were confirmed by experiments with either acetate- or glucose-limited chemostats. In chemostats in which both of the substrates were limiting, all of the enzymes displayed significant activities. Glucose 6-phosphate dehydrogenase activity was inhibited by acetyl coenzyme A and acetyl phosphate but not by acetate. It is proposed that diauxic growth is based on the control of enzymes involved in acetate or glucose dissimilation by which acetate or its metabolites control the expression and activity of glucose enzymes.     

Metabolic regulation in Pseudomonas oxalaticus OX1

Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and Q _{O 2}values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation appeared upon addition of formate. In the reciprocal transition, ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation rate declined sharply after formate exhaustion, and oxalyl-CoA reductase appeared only after addition of oxalate. The intracellular NAD and NADP concentrations measured in the same experiments are reported. At substrate exhaustion the proportion of NAD in the reduced form fell from 15–20% to 2%. On addition of formate to an oxalate-starved culture there was an immediate increase in the proportion of NADH to 50%; such an increase was not observed in the reverse experiment.Abbreviations RuDP ribulose-1,5-diphosphate - HEPES 2-(N-2 hydroxyethylpiperazin-N-yl) ethane sulphonic acid     

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO₂ or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [⁷⁵Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.Abbreviations DTE dithioerythritol - TES N-Tris (hydroxymethyl) methyl-2-amino-ethane sulfonic acid     

Diauxic growth in Azotobacter vinelandii.

Azotobacter vinelandii exhibited diauxie when grown in a medium containing both acetate and glucose as carbon sources. Acetate was used as the primary carbon source during the acetate-glucose diauxie. Uptake of acetate was constitutively expressed during both diauxic phases of growth. Induction of the glucose uptake system was inhibited in the presence of acetate. Acetate was also the preferred growth substrate for A. vinelandii grown in a medium containing either fructose, maltose, xylitol, or mannitol. The tricarboxylic acid cycle intermediates citrate, isocitrate, and 2-oxoglutarate inhibited glucose utilization in cells grown in glucose medium containing these substrates, and diauxic growth was observed under these growth conditions. Temporal expression of isocitrate-lyase, ATPase, and nitrogenase was exhibited during acetate-glucose diauxie.     

Diauxic growth of Clostridium thermosaccharolyticum on glucose and xylose

Abstract Clostridium thermosaccharolyticum growing on medium containing glucose and xylose exhibits classical diauxic growth in which glucose is utilized during the first phase. The lag period between growth phases is associated with induction of synthesis of a xylose transport system together with the enzymes xylose isomerase and xylukokinase. Xylose metabolism by this organism is therefore shown to be inducible and subject to repression by glucose. Xylose utilization by cells adapted to this carbon source is also prevented immediately upon addition of glucose to the culture, suggesting a direct inhibitory effect of glucose on xylose uptake or metabolism.     

New patterns of mixed-substrate utilization during batch growth of Escherichia coli K12

Microbial growth on mixtures of substrates is of considerable engineering and biological interest. Most of the work until now has dealt with microbial growth on binary mixtures of sugars or polyols. In these cases, it is often found that no matter how the inoculum is precultured, only one of the two substrates is consumed in the first growth phase, leading to the diauxic growth pattern. The goal of the experiments reported here is to investigate growth on mixtures containing at least one organic acid. These experiments show that the substrate utilization patterns in such mixtures are qualitatively different from the diauxic growth pattern. For instance, during growth of Escherichia coli K12 on certain binary mixtures of organic acids, the two substrates are utilized simultaneously, and the mixed-substrate maximum specific growth rate exceeds the single-substrate maximum specific growth rate on either one of the two constituent substrates. Furthermore, the very same mixed-substrate maximum specific growth and substrate uptake rates are observed no matter how the inoculum is precultured. On the other hand, in a mixture of glucose and pyruvate, the maximum specific growth rate seems to depend on the preculturing conditions, thus suggesting the existence of multiple physiological quasi-steady states. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 747-757, 1997.     

Regulation of the utilization of 4-hydroxybenzoate and 4-hydroxycinnamate in batch and continuous cultures of Pseudomonas testosteroni

Pseudomonas testosteroni metabolized 4-hydroxycinnamate by an initial cleavage of the side chain to yield acetate and the aromatic moiety, 4-hydroxybenzaldehyde. The latter was further oxidized via 4-hydroxybenzoate to protocatechuate, which underwent meta cleavage. During growth of the organism on 4-hydroxycinnamate, the for acetate showed an undulating pattern, which was attributed to alternating induction and repression of enzymes involved in the oxidation of acetate. Repression was caused either by 4-hydroxybenzoate or by its later metabolites, formate and pyruvate.In batch culture, P. testosteroni oxidized mixtures of 4-hydroxybenzoate and 4-hydroxycinnamate in a diauxic pattern. The capacity to oxidize 4-hydroxycinnamate appeared in the cells before 4-hydroxybenzoate was exhausted, indicating that the enzymes catalysing the conversion of 4-hydroxycinnamate into 4-hydroxybenzoate. were induced despite the presence of 4-hydroxybenzoate. The induction of these early enzymes of 4-hydroxycinnamate catabolism started when the molar concentration ratio of 4-hydroxybenzoate to 4-hydroxycinnamate fell below a value of 0.3.In continuous culture of P. testosteroni on a mixture of 4-hydroxybenzoate and 4-hydroxycinnamate, both substrates were almost completely utilized up to a dilution rate of about 0.5/h. At higher dilution rates, 4-hydroxycinnamate was decreasingly utilized so that eventually at a dilution rate of 0.74/h, its effluent concentration equalled its influent concentration. At D _M, a utilization ratio of 1.23 in favour of 4-hydroxybenzoate was found to become established in the culture. The of the cells for acetate was maximal at a dilution rate of 0.38/h and decreased before 4-hydroxycinnamate utilization was at its peak at 0.59/h. This suggested that it was mainly the aromatic moiety of 4-hydroxycinnamate which was metabolized at high dilution rates. The failure to utilize acetate at high dilution rates was apparently due to the repression of its catabolic enzymes by later metabolites of 4-hydroxybenzoate and to the relatively low concentration of acetate in the fermenter. This low concentration, due to the continuous washout of acetate, prevented it from relieving the repression.Abbreviations 4HB 4-hydroxybenzoate - 4HC 4-hydroxycinnamate - D _M dilution rate allowing maximal cell output rate - OD optical density     

Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui

Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Regulation of methanol oxidation and carbon dioxide fixation in Xanthobacter strain 25a grown in continuous culture

The regulation of C₁-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization (D=0.10 h^-1) of acetate and the C ₁-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by heterotrophic substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO₂ fixation via the Calvin cycle in Xanthobacter strain 25 a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO₂ fixation only occurs under conditions with a diminished supply of heterotrophic carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate.Abbreviations FDH formate dehydrogenase - FBPase fructose-1,6-bisphosphatase - ICDH isocitrate dehydrogenase - MDH methanol dehydrogenase - PQQ pyrrolo quinoline quinone - PRK phosphoribulokinase - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate - TCA tricarboxylic acid cycle     

Oxalyl-coenzyme A reduction to glyoxylate is the preferred route of oxalate assimilation in Methylobacterium extorquens AM1

Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and (13)C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions.     

Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose

Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By (13)C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol. (mg of protein)(-1). min(-1), respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol. [mg of protein](-1). min(-1)) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol. [mg of protein](-1). min(-1)). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol. [mg of protein](-1). min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.