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     

Metabolic regulation in Pseudomonas oxalaticus OX1. Diauxic growth on mixtures of oxalate and formate or acetate

Diauxic growth was observed in batch cultures of Pseudomonas oxalaticus when cells were pregrown on acetate and then transferred to mixtures of acetate and oxalate. In the first phase of growth only acetate was utilized. After the exhaustion of acetate from the medium enzymes involved in the metabolism of oxalate were synthesized during a lag phase of 2 h, followed by a second growth phase on oxalate. When the organism was pregrown on oxalate, oxalate utilization from the mixture with acetate completely ceased after a few hours during which acetate became the preferred substrate. Similar observations were made with formate/oxalate mixtures in which formate was the preferred substrate. Until formate was exhausted, it completely suppressed oxalate metabolism, again resulting in diauxic growth. However, when the organism was pregrown on oxalate and then transferred to mixtures of oxalate and formate, both substrates were utilized simultaneously although the initial rate of oxalate utilization from the mixture was strongly reduced as compared to growth on oxalate alone.Since both preferred substrates cross the cytoplasmic membrane by diffusion, whereas oxalate is accumulated by an inducible, active transport system, the effect of acetate and formate on oxalate transport was studied at different external pH values. At pH 5.5 both substrates completely inhibited oxalate transport. However, at pH 7.5, the pH at which the diauxic growth experiments were performed, formate and acetate did not affect oxalate transport. Growth patterns and enzymes profiles suggest that, at higher pH values, formate and acetate possibly affect oxalate utilization via an effect on the internal pool of oxalyl-CoA, the first product of oxalate metabolism.Abbreviations PMS phenazine methosulphate - RuBPCase ribulosebisphosphate carboxylase - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - p.m.f. protonmotive force     

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.     

Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Utilization of oxalate by cells after adaptation from growth on formate to growth on oxalate

1. The labelling patterns of phosphoglycerate obtained from formate-grown or oxalate-grown Pseudomonas oxalaticus after exposure for 15sec. to [¹⁴C]formate or [¹⁴C]oxalate respectively were determined. 2. The phosphoglycerate obtained from the formate-grown cells contained 78% of the radioactivity in the carboxyl group. This is in accord with that predicted for operation of the ribulose diphosphate cycle of carbon dioxide fixation. 3. The labelling pattern of the phosphoglycerate obtained from the oxalate-grown cells approached uniformity, as predicted for the heterotrophic pathway of oxalate assimilation. The departure from complete uniformity may have been due to concurrent ¹⁴CO₂ fixation into C₄ dicarboxylic acids. 4. The labelling pattern of phosphoglycerate obtained from cells that had just started to grow on oxalate after adaptation from formate was determined after incubation of the cells for 15sec. with [¹⁴C]oxalate. This pattern approached uniformity. 5. The pathway of incorporation of ¹⁴CO₂ into cells that had just started to grow on oxalate after adaptation from formate, in the presence of either formate or oxalate as energy source, was studied by chromatographic and radio-autographic analysis. 6. It is concluded from the isotopic data that a mixed heterotrophic–autotrophic metabolism, with the former mode predominating, operates in the initial stages of growth on oxalate after adaptation from growth on formate.     

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     

Microbial growth on oxalate by a route not involving glyoxylate carboligase

1. The metabolism of oxalate by the pink-pigmented organisms, Pseudomonas AM1, Pseudomonas AM2, Protaminobacter ruber and Pseudomonas extorquens has been compared with that of the non-pigmented Pseudomonas oxalaticus. 2. During growth on oxalate, all the organisms contain oxalyl-CoA decarboxylase, formate dehydrogenase and oxalyl-CoA reductase. This is consistent with oxidation of oxalate to carbon dioxide taking place via oxalyl-CoA, formyl-CoA and formate as intermediates, and also reduction of oxalate to glyoxylate taking place via oxalyl-CoA. 3. The pink-pigmented organisms, when grown on oxalate, contain l-serine–glyoxylate aminotransferase and hydroxypyruvate reductase but do not contain glyoxylate carboligase. The converse of this obtains in oxalate-grown Ps. oxalaticus. This indicates that, in contrast with Ps. oxalaticus, synthesis of C₃ compounds from oxalate by the pink-pigmented organisms occurs by a variant of the `serine pathway' used by Pseudomonas AM1 during growth on C₁ compounds. 4. Evidence in favour of this scheme is provided by the finding that a mutant of Pseudomonas AM1 that lacks hydroxypyruvate reductase is not able to grow on oxalate.     

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     

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     

Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.Abbreviations API appareils et procédés d'identification - CS citrate synthase - ED Entner-Doudoroff pathway - FBP fructose-1,6-bisphosphate - FDH formate dehydrogenase - HPS hexulose-6-phosphate synthase - ICDH isocitrate dehydrogenase - KDPG 2-keto-3-deoxy-6-phosphogluconate - MDH methanol dehydrogenase - PRK phosphoribulokinase - PQQ pyrrolo quinoline quinone - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate     

Substrate inhibition inPseudomonas oxalaticus OX1: a kinetic study of growth inhibition by oxalate and formate using extended cultures

Pseudomonas oxalaticus OX1 has been grown in a mineral salts medium with oxalate or formate as the sole source of carbon and energy. At concentrations of these substrates above 50mm inhibition of growth was indicated by a long and variable lag phase in batch culture. This inhibition was further studied by estimating maximum specific growth rates at different substrate concentrations using the extended culture technique for control of the substrate concentration. With formate, inhibition became apparent at substrate concentrations above 20mm, whereas oxalate inhibited growth at concentrations above 15mm. Complete inhibition was not observed even at concentrations of 100mm. A number of inhibition functions were fitted with the experimental data using computer analysis. The results indicated that the Haldane equation was the simplest function to describe quantitatively the kinetics of the observed substrate inhibition. Studies on the rate of oxygen uptake at different concentrations of oxalate indicated that respiration was much more sensitive to inhibition than growth. However with formate, inhibition of respiration was not observed up to concentrations of 50mm, indicating that different mechanisms may underlie the observed growth inhibition by the two substrates.     

Chloromethane Metabolism by Methylobacterium sp. Strain CM4

Methylobacterium sp. strain CM4 metabolized chloromethane quantitatively with a molar yield of 2.8 g of whole-cell protein/mol of C. This value was similar to that observed after growth with methanol (2.9 g of protein/mol of C) and about three times larger than the yield with formate (0.94 g of protein/mol of C). Chloromethane dehalogenation activity was inducible. MiniTn5 transposon insertion mutants with altered growth characteristics with chloromethane and other C₁ compounds were isolated and characterized. Nine of these were unable to grow with chloromethane but were able to grow with methanol, methylamine, or formate. Seventy-three transposon mutants that were defective in the utilization of either methanol, methylamine, methanol plus methylamine, or formate could still grow with chloromethane. Based on the protein yield data and the properties of the transposon mutants, we propose a pathway for chloromethane metabolism that depends on methyltransferase and dehydrogenase activities.     

Comparison of spiroplasmas by polyacrylamide gel electrophoresis of cell proteins

Pseudomonas oxalaticus utilized sodium acetate or fructose, in addition to sodium formate known to be assimilated via the reductive pentose phosphate pathway. The generation time during growth on fructose (2 h, 10 min) was considerably shorter than observed for other pseudomonads, which sequentially utilize a phosphoenolpyruvate-dependent phosphotransferase system and 1-phosphofructoninase during growth on fructose. In contrast, extracts prepared from fructose-grownP. oxalaticus contaned enzyme activities indicative of the Entner-Doudoroff pathway, while 1-phosphofructokinase was not found. Our studies indicate thatP. oxalaticus may be useful as a model organism for studies of CO₂ fixation.     

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.     

Oxalate utilisation is widespread in the actinobacterial genus Kribbella

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (^?OOC-COO^?) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.     

Clostridium difficile Is an Autotrophic Bacterial Pathogen

During the last decade, Clostridium difficile infection showed a dramatic increase in incidence and virulence in the Northern hemisphere. This incessantly challenging disease is the leading cause of antibiotic-associated and nosocomial infectious diarrhea and became life-threatening especially among elderly people. It is generally assumed that all human bacterial pathogens are heterotrophic organisms, being either saccharolytic or proteolytic. So far, this has not been questioned as colonization of the human gut gives access to an environment, rich in organic nutrients. Here, we present data that C. difficile (both clinical and rumen isolates) is also able to grow on CO₂+H₂ as sole carbon and energy source, thus representing the first identified autotrophic bacterial pathogen. Comparison of several different strains revealed high conservation of genes for autotrophic growth and showed that the ability to use gas mixtures for growth decreases or is lost upon prolonged culturing under heterotrophic conditions. The metabolic flexibility of C. difficile (heterotrophic growth on various substrates as well as autotrophy) could allow the organism in the gut to avoid competition by niche differentiation and contribute to its survival when stressed or in unfavorable conditions that cause death to other bacteria. This may be an important trait for the pathogenicity of C. difficile.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Formate-dependent autotrophic growth in Sinorhizobium meliloti

It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

Microbial growth on C1 compounds. The role of folate in the metabolism of Pseudomonas AM1

1. The growth of Pseudomonas AM1 is much more sensitive to inhibition by sulphanilamide when methanol, rather than succinate, acts as the sole carbon and energy source; a sulphanilamide concentration of 1mm, which causes almost complete inhibition of growth on methanol, has little effect in a succinate medium. 2. Similar results have been obtained with sulphadiazine and sulphathiazole. Sulphanilic acid has little effect. 3. A similar differential sensitivity to sulphanilamide is shown by Protaminobacter ruber and Pseudomonas extorquens when grown on methanol media as compared with succinate. 4. Sulphanilamide inhibited the growth of Pseudomonas oxalaticus on formate, oxalate and succinate media to about the same extent. 5. Inhibition of growth of Pseudomonas AM1 by sulphanilamide is accompanied by an accumulation of glycine in the cells. 6. Inhibition of growth by sulphanilamide can be reversed by p-aminobenzoic acid. 7. Microbiological assays of the folate content of Pseudomonas AM1 have been performed after growth on both methanol and succinate, and the results are discussed in terms of differences in metabolism.