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     

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.     

Active transport of oxalate by Pseudomonas oxalaticus OX1

Membrane vesicles isolated from oxalategrown cells of Pseudomonas oxalaticus accumulated oxalate by an inducible transport system in unmodified form against a concentration gradient. This accumulation was dependent on the presence of a suitable electron donor system such as ascorbate-phenazinemethosulphate. In the presence of this energy source, steady state levels of accumulation of oxalate were 10–20-fold higher than in its absence. The oxalate transport system involved showed a high affinity for oxalate (K _m =11 M) and was highly specific. Oxalate transport was not affected by the presence of other dicarboxylic acids, such as malate, succinate and fumarate and only partly inhibited by acetate. The energy requirement for oxalate transport is discussed and it is concluded that this requirement is most likely equivalent to 1 mole of ATP per mole of oxalate.Abbreviation PMS phenazinemethosulphate     

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. 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     

Regulation of autotrophic metabolism in Pseudomonas oxalaticus OX1 wild-type and an isocitrate-lyase-deficient mutant

In Pseudomonas oxalaticus the activity and synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are regulated by inactivation and endproduct repression, respectively. Phosphoenolpyruvate (PEP) has been suggested to function as a signal molecule for the latter control system. During growth of the organism in carbon-source-limited continuous cultures with various ratios of acetate and formate in the feed, the RuBisCO levels varied considerably, but no correlation was observed with the intracellular concentrations of PEP. To study whether the repression exerted by acetate utilization was dependent on the synthesis of glycolytic intermediates from this compound, an acetate-negative mutant defective in isocitrate lyase was isolated and characterized. Clear evidence was obtained that in this mutant acetate is as effective in repressing RuBisCO synthesis as in the wild-type. It therefore appears more likely that acetyl-CoA or a closely related metabolite functions as a signal molecule in the regulation of RuBisCO synthesis.     

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     

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     

Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1

A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides (OS1 and OS2): [structure: see text] where R=(S)-Pyr(-->4,6) in OS1 and alpha-Rha-(1-->3) in OS2. All sugars are D-pyranoses, except Rha, which is L-pyranose. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.     

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.     

The role of residue Thr249 in modulating the catalytic efficiency and substrate specificity of catechol-2,3-dioxygenase from Pseudomonas stutzeri OX1

Bioremediation strategies use microorganisms to remove hazardous substances, such as aromatic molecules, from polluted sites. The applicability of these techniques would greatly benefit from the expansion of the catabolic ability of these bacteria in transforming a variety of aromatic compounds. Catechol-2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1 is a key enzyme in the catabolic pathway for aromatic molecules. Its specificity and regioselectivity control the range of molecules degraded through the catabolic pathway of the microorganism that is able to use aromatic hydrocarbons as growth substrates. We have used in silico substrate docking procedures to investigate the molecular determinants that direct the enzyme substrate specificity. In particular, we looked for a possible molecular explanation of the inability of catechol-2,3-dioxygenase to cleave 3,5-dimethylcatechol and 3,6-dimethylcatechol and of the efficient cleavage of 3,4-dimethylcatechol. The docking study suggested that reduction in the volume of the side chain of residue 249 could allow the binding of 3,5-dimethylcatechol and 3,6-dimethylcatechol. This information was used to prepare and characterize mutants at position 249. The kinetic and regiospecificity parameters of the mutants confirm the docking predictions, and indicate that this position controls the substrate specificity of catechol-2,3-dioxygenase. Moreover, our results suggest that Thr249 also plays a previously unsuspected role in the catalytic mechanism of substrate cleavage. The hypothesis is advanced that a water molecule bound between one of the hydroxyl groups of the substrate and the side chain of Thr249 favors the deprotonation/protonation of this hydroxyl group, thus assisting the final steps of the cleavage reaction.     

PHK from phenol hydroxylase of Pseudomonas sp. OX1. Insight into the role of an accessory protein in bacterial multicomponent monooxygenases

Bacterial multicomponent monooxygenases (BMMs) are members of a wide family of diiron enzymes that use molecular oxygen to hydroxylate a variety of aromatic compounds. The presence of genes encoding for accessory proteins not involved in catalysis and whose role is still elusive, is a common feature of the gene clusters of several BMMs, including phenol hydroxylases and several soluble methane monooxygenases. In this study we have expressed, purified, and partially characterized the accessory component PHK of the phenol hydroxylase from Pseudomonas sp. OX1, a bacterium able to degrade several aromatic compounds. The phenol hydroxylase (ph) gene cluster was expressed in Escherichia coli/JM109 cells in the absence and in the presence of the phk gene. The presence of the phk gene lead to an increase in the hydroxylase activity of whole recombinant cells with phenol. PHK was assessed for its ability to interact with the active hydroxylase complex. Our results show that PHK is neither involved in the catalytic activity of the phenol hydroxylase complex nor required for the assembly of apo-hydroxylase. Our results suggest instead that this component may be responsible for enhancing iron incorporation into the active site of the apo-hydroxylase.